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FOCUS
PUBLISHED FOR THE LIFE SCIENTIST BY LIFE TECHNOLOGIES, INC.

VOLUME 21 NUMBER 3 1999

Featured Papers:
Transfection in 96well Plates 58 Mammalian Cell Lines for Transfection 62 Fluorescent DNA Ladders 64 Helpful Tips for Custom Primers 69 Some Basics of Cell Culture Media 76

c o n t e n t s

ABOUT THE COVER: Transfection in 96-well plates. (See page 58).

FOCUS
TRANSFECTION
Jean-Pierre Pichet and Valentina Ciccarone Linda Roy, Sharon Cates, Kevin Schifferli, Jean-Pierre Pichet, Valentina Ciccarone, Shelley Bennett, and Pamela Hawley-Nelson

FOCUS contains manuscripts describing novel techniques, improvements of common techniques, simplied protocols, and troubleshooting.Instructions to Authors are available on the Internet or from the editor: FOCUS Editor: Dr. Doreen Cupo Life Technologies, Inc. 9800 Medical Center Drive Rockville, MD 20849-6482 (800) 828-6686 (301) 610-8000 outside the U.S. E-mail: dcupo@lifetech.com Assistant to the Editor: Karen Carstensen Salovich Copyright Life Technologies, Inc., 1999 FOCUS is published triannually by Life Technologies, Inc. POSTMASTER: Send address changes to FOCUS, Life Technologies, Inc. P.O. Box 6482, Rockville, MD 20849-6482 Requests for subscriptions and address changes should be directed to the nearest Life Technologies ofce: U.S. ACADEMIC ORDERS To Order/TECH-LINE: SM (800) 828-6686 Fax: (800) 331-2286 U.S. GOVERNMENT ORDERS To Order: (888) 584-8929 Fax: (888) 584-8930 U.S. INDUSTRIAL ORDERS To Order/TECH-LINE: (800) 874-4226 Fax: (800) 352-1468 Internet www.lifetech.com info@lifetech.com INTERNATIONAL ORDERS U.S.A. Ofce for Latin America To Order/TECH-LINE: (301) 610-8709 Fax: (301) 610-8724 AUSTRALIA Melbourne To Order/TECH-LINE: 1800 331 627 Tel: (03) 9558 9622 Fax: (03) 9558 9722 CANADA Burlington, Ontario To Order: (800) 263-6236 TECH-LINE: (800) 757-8257 Fax: (800) 387-1007 EUROPE Paisley, Scotland To Order: 0800 269210 TECH-LINE: 0800 838380 Fax: 0800 243485 HONG KONG Tsuen Wan Tel: (852) 2407-8450 Fax: (852) 2408-2280/2409-6043 INDIA New Delhi To Order: 91-11-577-3282 Fax: 91-11-577-3281 JAPAN Tokyo Tel: (81-3) 3663-8241 Fax: (81-3) 3663-8242 NEW ZEALAND Auckland To Order/TECH-LINE: 0800 600 200 Tel: (09) 579 3024 Fax: (09) 579 3119 PEOPLES REPUBLIC OF CHINA Beijing To Order: (86-10) 6256-3836 Fax: (86-10) 6256-4852 PEOPLES REPUBLIC OF CHINA Shanghai To Order: (86-21) 6471-1313 Fax: (86-21) 6471-1313 TAIWAN Taipei Tel: (886-2) 2651-6156 Fax: (886-2) 2653-8100 Editorial Review Board: Holly Anderson, Paul Battista, Stephen Goren, James Hartley, Curtis Henrich, Larry Mertz, Frank Swartzwelder Contributing Editorial Reviewers: J.J. Lin, Dave Schuster

Transfection of Mammalian Cells in 96-Well Plates with LIPOFECTAMINE 2000 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Cationic Lipid Reagent Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 High Transfection Efciency of Cloned Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

ELECTROPHORESIS

DNA Ladders for Fluorescent Fragment Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Heather Jordan and Joseph Solus

PCR

One-Step RT-PCR to Detect Cytokine/Chemokine Induction in Macrophages . . . . . . . . . . . . . . . . . 66

Monique Bongers, Ekke Liehl, and Johannes Barsig

Helpful Tips for Custom Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Technical Services

AGRICULTURAL BIOTECHNOLOGY

AFLP Analysis of the Fruit Fly Ceratitis capitata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Gino Corsini, Augusto Manubens, Manuel Lladser, Sergio Lobos, Daniela Seelenfreund, and Carlos Lobos

MOLECULAR BIOLOGY

Buffer Compatibility for Common Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Joe Crouse, Teresa Myers, and Julie Brent

CELL CULTURE

How Basal Media Provide an Optimal Growth Environment for Cell Culture . . . . . . . . . . . . . . . . 76
Kevin Grady

AFLP is a trademark of Keygene n.v. CFLP is a trademark of Third Wave Technologies, Inc. GeneScan is a registered trademark of The PerkinElmer Corporation, Inc. Kodak is a registered trademark of Eastman Kodak Co. Triton is a registered trademark of Rohm & Haas, Co. TRIZOL is a registered trademark of Molecular Research Center, Inc. Tween 20 is a registered trademark of ICI Americas, Inc. Purchase of Taq DNA Polymerase is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research and development in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. This product is sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., and The Perkin-Elmer Corporation Inc.
99-142 Part No. 53077

Printed on recycled paper

Essential Technologies for the Science of Life

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Jean-Pierre Pichet Valentina Ciccarone Molecular Biology Research and Development Life Technologies, Inc. Rockville, Maryland 20849

Transfection of Mammalian Cells in 96-Well Plates with

LIPOFECTAMINE 2000 Reagent

IPOFECTAMINE 2000 Reagent

Panel A 293-H Cells

320 18,000 16,000 DNA (ng) 240 ng -gal/cm2 14,000 12,000 10,000 8,000 6,000 4,000 0.2 0.4 0.6 0.8 1.0 1.2 2,000 0 0.2 0.4 160

Panel B

DNA (ng)

160

ng -gal/cm2

transfects cells at very high efciencies, resulting in high levels of recombinant protein expression (1). The protocol for transfection of cells with LIPOFECTAMINE 2000 Reagent involves very few steps and can be performed in serum-containing medium. Therefore, this new reagent is especially suited for transfections in the 96-well format, which is ideal for high-throughput protocols. In this report, we optimized transfection conditions for 96-well plates with the 3 cell lines commonly used in high-throughput protocolsCHO, COS, and 293using either a "Standard Protocol," where cells are plated the day before transfection, or a "One-day Protocol," where a cell suspension is added to the DNA-reagent complexes prepared in the wells. The sensitivity of these protocols to measure gene activity in cDNA libraries is also shown.

80

LIPOFECTAMINE 2000 Reagent (l)

0.6

0.8

1.0

1.2

320 240 160 DNA 80 (ng)

LIPOFECTAMINE 2000 Reagent (l)

COS-7L Cells
320 3,000 2,500 240 2,000 1,500 1,000 500 0.2 0.4 0.6 0.8 1.0 1.2 0 LIPOFECTAMINE 2000 Reagent (l) 0.2 0.4 0.6 0.8 1.0 1.2

METHODS
CELL CULTURE. All cell lines, culture media, sera, and reagents were from Life Technologies. CHO-S (Cat. No. 11619), COS-7L (Cat. No. 11622), and 293-H (Cat. No. 11631) cells (2) were cultured in Dulbeccos MEM (D-MEM High Glucose: 4,500 mg/L D-glucose, with 584 mg/L Lglutamine and 15 mg/L phenol red) supplemented with 0.1 mM nonessential amino acids (NEAA) and 10% FBS. Cell cultures were maintained at 37C in a humidied, 5% CO2 incubator. For COS and 293 cells, poly-D-Lysine coated plates were used for transfection protocols. TRANSFECTION. For the Standard Protocol, cells were plated in 96-well plates the day before transfection in 100 l of growth medium. Several seeding densities were tested for each cell type (CHO-S, 2 104 to 4 104; COS-7L, 1.5 104 to 2.5 104; 293-H, 4 104 to 6 104 cells/well). 58

80 320 240 160 DNA 80 (ng)

LIPOFECTAMINE 2000 Reagent (l)

CHO-S Cells
320 4,500 4,000 DNA (ng) 240 ng -gal/cm2 3,500 3,000 2,500 2,000 1,500 1,000 500 0.2 0.4 0.6 0.8 1.0 1.2 0 LIPOFECTAMINE 2000 Reagent (l) 0.2 0.8 1.0 1.2 LIPOFECTAMINE 2000 Reagent (l) 0.4 0.6 320 240 160 DNA 80 (ng) 160

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FIGURE 1. Transfection of cells using the standard protocol. Cells were transfected with pCMVSPORT-gal DNA and LIPOFECTAMINE 2000 Reagent. At 24 h after complex addition, duplicate plates were assayed with X-gal (panel A) and ONPG (panel B). Results from the optimal seeding density for each cell line are shown: 2 104 for CHO-S, 2.5 104 for COS-7L, and 5 104 for 293-H.

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pCMVSPORT-gal (Cat. No. 19586) and pCMVSPORT6-gal-neo plasmid DNA were puried using the CONCERT High Purity Plasmid Purication Maxiprep System (Cat. No. 11452). For each well, DNA and LIPOFECTAMINE 2000 Reagent (Cat. No. 11668) were rst diluted separately in 25 l OPTI-MEM I Reduced Serum Medium without serum. The diluted LIPOFECTAMINE 2000 Reagent was incubated for 5 min at room temperature, before mixing with the diluted DNA. The DNA-reagent complexes, in 96-well plates, were incubated at room temperature for 20 min for complex formation. Using duplicate plates for each cell line, 50 l of complex was added directly to the cells in their growth medium and gently mixed. For the rapid, One-day Protocol, DNA-reagent complexes were prepared as described above in 96-well plates. Cells were trypsinized, and a 100-l cell suspension was added to the complexes in wells. CDNA LIBRARY SCREEN. pCMVSPORT6gal-neo was serially diluted into total plasmid DNA from a human brain cDNA library. The total DNA amount transfected was constant in all wells at 320 ng. The amount of pCMVSPORT6-gal-neo DNA in the wells varied from 156 pg to 320 ng. The transfection was performed with the Standard Protocol. -GAL ACTIVITY. At 24 h post-transfection, the cells were rinsed with D-PBS and either xed and stained in situ with X-gal (3) or lysed and harvested in 150 l of 0.1 M Tris-HCl (pH 8.0), 0.1% Triton X 100 to measure -gal enzymatic activity using ONPG (4). Cells were frozen in lysis buffer at 80C for at least 1 h prior to assay. RESULTS AND DISCUSSION STANDARD PROTOCOL. To determine optimal conditions for transfection in a 96-well plate, LIPOFECTAMINE 2000 Reagent and DNA
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Cell Line CHO-S COS-7L 293-H

Seeding Density (cells/well) 2 104 2.5 104 5 104

DNA (ng/well) 240 320 320

LIPOFECTAMINE 2000 Reagent (l/well) 1 1 1

TABLE 1. Optimal transfection conditions of cells in 96-well plates using the standard protocol. Cell Line CHO-S COS-7L 293-H Seeding Density (cells/well) 5 104 6 10
4

DNA (ng/well) 320 320 320

LIPOFECTAMINE 2000 Reagent (l/well) 0.8 0.60.8 0.4

1.2 105

TABLE 2. Optimal transfection conditions of cells with the rapid, one-day protocol.

293-H Cells 18,000 16,000 14,000 12,000 ng -gal/cm2 10,000 8,000 6,000 4,000 2,000 0 0.2 0.4 500 320 240 160 DNA 80 (ng) 0 0.2 0.4 ng -gal/cm2 3,000 2,500 2,000 1,500 1,000

COS-7L Cells

0.6

0.8

2.0

0.6

1.2

0.8

2.0

1.2

320 240 160 DNA 80 (ng)

LIPOFECTAMINE 2000 Reagent (l)

LIPOFECTAMINE 2000 Reagent (l)

FIGURE 2. Transfection of cells using the rapid, one-day protocol. DNA-LIPOFECTAMINE 2000 Reagent complexes were prepared in 96-well plates and cell suspension added to each well. At optimal conditions, the cell concentrations were 1.2 105 cells/well for 293-H cells and 6 104 cells/well for COS-7L cells in a total volume of 100 l of growth medium.

concentration were evaluated at 3 cell densities. Good transfection efciencies were obtained with the 3 cell lines, and optimal ranges were identied (gure 1, table 1). ONE-DAY PROTOCOL. A more rapid, One-day Protocol where the complexes are prepared in a 96-well plate and a cell suspension is added directly to the complexes was evaluated. In this protocol, it is not necessary to plate the cells the day before transfection. Ranges of DNA and reagent concentrations at 3 cell densities were evaluated to determine optimal conditions. The optimal cell

concentration for the rapid, One-day Protocol was 2.5 times higher than for the Standard Protocol (gure 2, table 2). The maximum activity for 293 and COS-7 cells was lower than with the Standard Protocol (40% for 293 and 50% for COS-7). However, the expression levels were good, and time savings may provide an advantage in some applications. For CHO cells, the activity was 70% lower, so this rapid protocol may not be as useful with this cell line. CDNA LIBRARY SCREENING. The standard protocol was used to determine the sensitivity
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Transfection of Mammalian Cells continued

Panel A COS-7L Cells
6,000

Panel B

5,000

4,000 ng -gal/cm2

3,000

2,000

1,000

0 320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156 320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156

Target DNA (ng)

Target DNA (ng)

293-H Cells
12,000

10,000

8,000 ng -gal/cm2

6,000

4,000

2,000

0 320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156 320 160 80 40 20 10 5 2.5 1.25 0.625 0.131 0.156 Target DNA (ng) Target DNA (ng)

FIGURE 3. Detection of gene expression in a cDNA library. Cells plated at their optimal seeding density were transfected with serially diluted pCMVSPORT6-gal-neo added to plasmid DNA from a cDNA library. The total amount of DNA transfected was always 320 ng. Wells in each column of the plate contained the same amount of pCMVSPORT6-gal-neo DNA. Duplicate plates were assayed with X-gal (panel A) and ONPG (panel B). Data in panel B are the mean SD for N = 8.

of detection of gene expression in a cDNA library. Expression was detected with small amounts of -gal plasmid (gure 3), suggesting that this protocol may be used for screening to detect specic gene expression in cDNA libraries. Detection levels will depend on the vector used and the assay sensitivity. For example, the better COS-7L sensitivity was due to the SV40 origin of replication in the plasmid allowing higher expression levels in the SV40-transformed COS-7L cells. Other detection methods (i.e. luminescence) may allow greater sensitivity.
60

In summary, LIPOFECTAMINE 2000 Reagent is highly efcient for transfection of cells in a 96-well format. Starting with the protocols described here, transfection conditions can be easily established for automated or robotic systems in highthroughput screening applications. ACKNOWLEDGEMENTS The authors thank Dr. Ray Hadley and Dr. Chris Gruber for the brain library cDNA. FOCUS

REFERENCES
1. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999) FOCUS 21, 54. Roy, L., Cates, S., Schifferli, K., Pichet, J.P., Ciccarone, V., Bennett, S., and Hawley-Nelson, P. (1999) FOCUS 21, 62. Sanes, J.R., Rubenstein, L.R., and Nicolas, J.F. (1986) EMBO J. 5, 3133. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York. Second Edition, p. 16.66.

2.

3. 4.

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Cationic Lipid Reagent Selection

W
Cell Line 293-F 293-H BE(2)C BHK-21 CHO-K1

hen starting transfections in your lab, the general guidelines to follow in selecting a reagent are summarized at the right. The table below contains recommended cationic lipid reagents for high-efciency transfection of key cell lines.* Please consult our web site (www.lifetech.com) for the most current additions to our recommended reagent table.

Application Difcult-to-transfect cells Most adherent cells Adherent cells in the presence of serum Suspension cells Insect cells Endothelial cells RNA Oligonucleotides

Recommended Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent or LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent DMRIE-C Reagent CELLFECTIN Reagent LIPOFECTIN Reagent DMRIE-C Reagent LIPOFECTIN Reagent, LIPOFECTAMINE Reagent, or CELLFECTIN Reagent

Mammalian Cell Type Human kidney Human kidney Human neuroblastoma Hamster kidney Hamster ovary Hamster ovary Hamster ovary Monkey kidney Monkey kidney Human primary passaged Human cervical cancer Human colon cancer Human brosarcoma Human endothelial Human endothelial Human primary passaged Dog kidney Human lung Mouse broblasts Rat pheochromocytoma Human breast cancer Monkey kidney Insect Cell Type Drosophila melanogaster Spodoptera frugiperda Spodoptera frugiperda

Recommended Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent DMRIE-C Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTIN with PLUS Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent (with serum) LIPOFECTIN Reagent LIPOFECTIN Reagent LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (with serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent (without serum) Recommended Reagent CELLFECTIN Reagent CELLFECTIN Reagent CELLFECTIN Reagent

CHO-S (adherent) CHO-S (suspension) (In CD-CHO Medium) COS-1 COS-7L Fibroblasts HeLa HT-29 HT-1080 HUAEC (primary) HUVEC (primary) Keratinocytes (In Keratinocyte-SFM) MDCK MRC-5 NIH-3T3 PC-12 SK-BR3 Vero Cell Line D.Mel-2 Sf9 Sf21

* Protocols for these transfections are in the TECH-ONLINE section of our web site at www.lifetech.com and available from the TECH-LINE.

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Linda Roy, Sharon Cates, Kevin Schifferli, Jean-Pierre Pichet, Valentina Ciccarone, Shelley Bennett, and Pamela Hawley-Nelson Research and Development Life Technologies, Inc. Rockville, Maryland 20849

Transfection High Cell Lines Efciency of Cloned

hinese Hamster Ovary (CHO) (1) cells, COS-7 (2) cells, and HEK 293 (3) cells are among the most commonly used mammalian cell lines for transfection, expression, and large-scale production of recombinant proteins. While these cell lines have been available for many years, the commercial lines have not always been easily transfected. Since these immortalized cell lines are aneuploid, sublines have evolved in various laboratories that are subtly different from the cells originally isolated, cultured, and banked at American Type Culture Collection (ATCC) or other cell banks. As a result, variability in transfection efciency has been observed. Some researchers have selected for more easily transfected variants. Life Technologies has used several better-transfecting sublines to isolate clones exhibiting high transfection efciency. In this paper, we show that these cell lines exhibit higher transfection activity than other available cells.

METHODS
CELL CULTURE. All cell culture media, sera,

and reagents were from Life Technologies unless otherwise noted. For transfection, cells were grown as adherent cultures in Dulbeccos MEM (D-MEM, high glucose: 4,500 mg/L D-glucose, with 584 mg/L L-glutamine and 15 mg/L phenol red) supplemented with 0.1 mM non-essential amino acids (NEAA) and 10% FBS at 37C in 5% CO2. ATCC cells were used <10 passages after thawing. We recommend Life Technologies cells for up to 30 passages or 3 months post-thaw. After many passages, the transfection efciency may change (9). Life Technologies cell lines used were 293-H (Cat. No. 11631); 293-F (Cat. No. 11625); CHO-S (Cat. No. 11619); and COS-7L (Cat. No. 11622). The parental lines were obtained as follows: 293-H (from Leaf Huang at the University of Pittsburgh), 293-F (from Robert Horlick at Pharmacopoeia), CHO-S (from Robert Tobey at Los Alamos), and COS-7L (from Tom Livelli at Specialty Media).
TRANSFECTION. The day before the transfection, 1.5 105 CHO cells, 2.0 105 HEK 293 cells, and 8.0 104 COS-7 cells were plated in 0.5 ml of growth medium in each well of 24-well plates. Since 293 and COS-7 cells are weakly adherent cells, they were plated on poly-D-lysine (50 g/well) coated plates. The cells were transfected with pCMVSPORT-gal DNA using LIPOFECTAMINE 2000 Reagent (Cat. No. 11668) or LIPOFECTAMINE PLUS Reagent according to manufacturers recommendations. The DNA was prepared using the CONCERT High Purity Maxiprep System. For transfection with LIPOFECTAMINE PLUS Reagent (4), the DNA was precomplexed with the PLUS Reagent by diluting 0.8 g DNA to 17 l with D-MEM/

NEAA and adding 8 l PLUS Reagent. The precomplexes were incubated at room temperature for 15 min. Meanwhile, 0 to 5 l of LIPOFECTAMINE Reagent were diluted to 25 l with D-MEM/NEAA and incubated for 5 min at room temperature. The DNA/PLUS Reagent was mixed with the diluted LIPOFECTAMINE Reagent and incubated for 15 min at room temperature. The growth medium was removed from the cells and replaced with 500 l of D-MEM/NEAA. The complexes were added to the cells and incubated for 4 h in 37C, 5% CO2. 500 l of D-MEM/NEAA containing 20% FBS were added to the cells. The transfections with LIPOFECTAMINE 2000 Reagent (5) were performed in growth medium (D-MEM/NEAA/10% FBS). 0 to 6 l of LIPOFECTAMINE 2000 Reagent were diluted to 50 l with DMEM/NEAA. DNA (0.8 g) was diluted to 50 l with D-MEM/NEAA. After 5 min, the diluted DNA was added to the diluted LIPOFECTAMINE 2000 Reagent and incubated for 15 min at room temperature. The complexes (100 l) were added to each well, and the cells were placed at 37C in 5% CO2.
b-GAL ACTIVITY. At 24 h after addition of complexes, cells were rinsed once with Dulbeccos Phosphate Buffered Saline Solution and lysed in 0.1 M Tris-HCl (pH 8.0), 0.1% Triton X-100 (v/v) and frozen at 70C overnight. Cell extracts were thawed and assayed for -gal activity using ONPG (7). Protein was determined by a Bradford assay. Alternatively, cells were xed and stained in situ with X-gal (8). Stained cells were photographed using a 10X objective on a Nikon inverted microscope with Hoffman optics. Percent stained cells was determined by counting 3 elds and averaging.

FIGURE 1. Expression of -gal. 293 cells from Life Technologies (panel A) or ATCC (panel B) were transfected using LIPOFECTAMINE 2000 Reagent and assayed after 24 h. The cells photographed are the peak activity from a dose response.

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Cell Line COS-7 COS-7L 293 293-F 293-H CHO-K1 CHO-S

Source ATCC Life Technologies ATCC Life Technologies Life Technologies ATCC Life Technologies

Transfection Efciency (% stained cells) LIPOFECTAMINE PLUS LIPOFECTAMINE 2000 Reagent Reagent 35 2 74 6 39 9 99 0 99 0 45 3 97 2 73 11 99 0 73 8 99 0 99 0 86 2 96 3

REFERENCES
1. 2. 3. 4. Puck, T. (1958) J. Exp. Med. 108, 945. Glutzman, Y. (1981) Cell 23, 175. Graham, F.L. (1977) J. Gen. Virol. 36, 319. Shih, P.-J., Evans K., Schifferli, K., Ciccarone, V., Lichaa, F., Masoud, M., Lan, J., and Hawley-Nelson, P. (1997) Focus 19, 52. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999) Focus 21, 54. Schifferli, K., Jessee, J., and Ciccarone, V. (1999) Focus 21, 16. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York. Second Edition, p. 16.66. Sanes, J.R., Rubenstein, L.R., and Nicholas, J.F. (1986) EMBO J. 5, 3133. Hawley-Nelson, P. and Shih, P.-J. (1995) Focus 17, 60.

5.

6. 7.

TABLE 1. Transfection efciency. Cells were stained for -gal at 24 h after addition of complexes.

8. 9.
160 140 120 ng -Gal/g protein ng -Gal/g protein 100 80 60 40 20 0 293-F 293-H HEK 293 FIGURE 2. Comparison of -gal activity. Peak transfection activity of Life Technologies () or ATCC () cell lines was determined with ONPG 24 h after addition of DNA/LIPOFECTAMINE 2000 Reagent complexes. 293 ATCC 45 40 35 30 25 20 15 10 5 0 CHO COS

The Help Box from Your Technical Support & Training Team Q. How does LIPOFECTAMINE 2000 Reagent improve transfection compared to other reagents? A. Higher levels of protein expression have been observed in most of the cell lines tested. In addition, the streamlined protocol allows the addition of complexes directly to cells without changing media. Q. Why do you recommend using medium without antibiotics during transfection? A. While penicillin and streptomycin are not toxic to eukaryotic cells, the increased cell permeability occurring during transfection leads to much higher levels of antibiotics getting into cells. This can lead to higher cell death. Q. Can I use the same amount of any cationic lipid reagent for my cell line? A. No. Optimize the amount of each reagent. Q. What is the shelf life of cationic lipid reagents? A. Stored at 4C in a closed container, they are stable for 12 months. Do not freeze the reagents. Q. Is it necessary to use medium without serum during transfection? A. No. It is essential to form the DNA/cationic lipid reagent complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serumcontaining medium.

RESULTS AND DISCUSSION Cell lines were evaluated for transfection efciency using 2 reagents. Results indicated that the clones selected for transfectability had high peak transfection efciency (gure 1, table 1). The magnitude of difference from cells not selected for transfection ability was affected by the transfection reagent, with LIPOFECTAMINE 2000 Reagent demonstrating >95% transfected cells for the Life Technologies cells. This high efficiency was possible with transfection in the presence of serum with LIPOFECTAMINE 2000 Reagent. Similarly, the peak -gal activity was higher in the clones selected for transfection (gure 2). In contrast to the percent of
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cells transfected, the CHO-S clone demonstrated almost twice the -gal activity of the CHO-K1 cell line. In summary, the CHO-S, 293-H, 293-F, and COS-7L cells were transfected better than ATCC cell lines. For added exibility, these cells are provided adapted to serumfree medium and suspension culture. This facilitates use of these cells for large-scale protein production after selection of a stably transfected clone. For CHO-S cells, it also allows transient transfection in suspension culture and the chemically dened CDCHO Medium (6). FOCUS

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Heather Jordan and Joseph Solus Research and Development Life Technologies, Inc. Rockville, Maryland 20849

DNA Ladders for Fluorescent Fragment Analysis

luorescence-based DNA electrophoresis systems are used often in genotyping applications to analyze polymorphic markers whose alleles differ by discrete size increments. To determine the sizes of the unknown fragments, the instruments and software usually require that the size standard be electrophoresed in the same lane or capillary with each sample.

b 500 490 480 460

b 500 490 470 450

440 430 420 410 400 390 380 370 360 350 340 320 300 280 270 260 250 240 230 220 200 180 170 160 150 140 130 120 110 100 90 80 60 50 FIGURE 1. The GENOTYPE DNA Ladders. Alternating lanes of a 4.25% denaturing polyacrylamide gel were loaded with the GENOTYPE TAMRA 60-500 DNA Ladder and the GENOTYPE TAMRA 50-500 DNA Ladder. 90 210 200 190 310 300 290 350 330

For gel-based systems, sharkstooth combs are used for higher sample throughput and easier loading of the thin gels. For the software to distinguish adjacent lanes from one another, it becomes necessary to load every other lane of the gel, electrophorese the samples briey, and load the remaining lanes of the gel. If all lanes of the gel were loaded at the same time, the DNA bands of the sizing ladder would appear as continuous lines across the entire gel and adjacent lanes would not be distinguishable. An alternative to time-consuming multiple gel loadings is to use DNA ladders with different band sizes in adjacent lanes. This paper describes uorescently labeled DNA ladders that can be used individually or as a dual-ladder conguration for simplied loading of gels with sharkstooth combs. METHODS For gel electrophoresis, the GENOTYPE TAMRA 50-500 DNA Ladder (Cat. No. 11726) and the GENOTYPE TAMRA 60-500 DNA Ladder (Cat. No. 11727) were analyzed on the ABI 377XL
5,000

Automated Sequencer and were used to determine the size of PCR products amplied from dinucleotide repeat microsatellite markers using primer sets from Genethon published sequences as previously described (1). For analysis, 1.5 l of sample or water were mixed with 2.75 l of deionized formamide, 0.25 l of DNA ladder, and 0.5 l of blue dextran solution (50 mg/ml blue dextran, 10 mM EDTA). The mixtures were heat denatured at 90C for 2 min, cooled on ice, and 1.5 l were electrophoresed on a 36-cm, 4.25% (GEL-MIX 4.25FA, Cat. No. 10773) polyacrylamide sequencing gel using a 36-lane sharkstooth comb. Data were analyzed using GeneScan 3.1.1 software. For capillary electrophoresis, the ladders were analyzed on a POP-6 matrix under denaturing conditions using an ABI 310 Capillary Genetic Analyzer. 0.25 l of the DNA ladder was mixed with 12 l of deionized formamide, heat denatured at 95C for 5 min, cooled on ice, and the entire sample injected. Data were analyzed using GeneScan 3.1.1 software.

R2 = 0.9994 4,500 4,000 3,500 Data Point 3,000 2,500 2,000 1,500 1,000

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FIGURE 2. Linear correlation of the GENOTYPE TAMRA 50-500 DNA Ladder. The GENOTYPE TAMRA 50-500 DNA Ladder was analyzed by electrophoresis on an ABI 377 gel. The uorescent fragments were detected as they passed by the scanning laser during electrophoresis. The "Data Point" (Y axis) is a measure of the number of scans across the gel by the laser prior to detection of the fragment and thus corresponds to the migration rate of the fragment.

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RESULTS AND DISCUSSION The GENOTYPE TAMRA DNA Ladders were analyzed in the dual-ladder conguration (gure 1). All lanes were loaded simultaneously and the 2 distinct band patterns were visible, allowing automated, error-free lane tracking by the software. The migration of bands was linear with respect to fragment size (gure 2). Several dinucleotide repeat microsatellite markers amplied from human DNA as previously described (1) were sized with the G ENO TYPE DNA Ladders. The measured sizes calculated using the GENOTYPE DNA Ladders (table 1) were consistent with data obtained previously using commercially available ladders (data not shown). The GENOTYPE TAMRA DNA Ladders were analyzed by capillary electrophoresis (gure 3). The electropherograms illustrate the wide size range and even peak intensities of the ladders. Orientation markers (doublets and triplets) of the GENOTYPE DNA Ladders are indicated by arrows. In summary, uorescent GENOTYPE DNA Ladders provide a dual-ladder format to facilitate automated lane tracking and eliminate multiple gel loadings. The ladders accurately sized human DNA markers using gel-based or capillary-based (data not shown) electrophoresis systems. EDITORS NOTE: The ladders are also available labeled with ROX (GENOTYPE ROX 50-500 DNA Ladder, Cat. No. 11728, and GENOTYPE ROX 60-500 DNA Ladder, Cat. No. 11729). FOCUS REFERENCE
1. Jordan, H., Darer, M., and Solus, J. (1999) FOCUS 21, 1.

Locus D1S196 D1S220 D1S234 D1S235 D9S167 D16S671 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1

Expected Size Range (bp) 267297 231251 226238 175195 260286 338372

Calculated Size (bp) GENOTYPE TAMRA GENOTYPE TAMRA 50-500 DNA Ladder 60-500 DNA Ladder 272.05 273.13 245.35 247.31 230.94 232.91 173.90 174.82 264.72 272.70 369.08 272.12 273.13 245.46 247.46 230.99 232.88 173.77 174.79 264.90 272.70 369.72

TABLE 1. Size determination of dinucleotide repeat microsatellite markers. Markers were amplied using Genethon published sequences, and the sizes of the amplicons were determined using the GENOTYPE TAMRA DNA Ladders on an ABI 377XL DNA Sequencer. The expected size range was determined by Genethon from analysis of a large number of alleles for each locus, but may not account for all alleles in the human population.

Size (b) 50 100 150 200 250 300 350 400 450 500

Fluorescence Intensity

1,200 800 400 0 GENOTYPE TAMRA 50500 DNA Ladder

1,200 800 400 0 GENOTYPE TAMRA 60500 DNA Ladder

FIGURE 3. Capillary electrophoresis of GENOTYPE TAMRA DNA Ladders.

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Monique Bongers, Ekke Liehl, and Johannes Barsig Novartis Research Institute Brunner Str. 59 1235 Vienna, Austria e-mail: monique.bongers@pharma.novartis.com

One-Step RT-PCR to Detect in Cytokine/Chemokine Induction

Macrophages
ABSTRACT The macrophage is a central cell in inammation, host defense, and wound healing. Macrophages are active as phagocytes and produce mediators that attract other leukocytes and/or initiate reconstruction of tissue lesions. We describe methods to study mediator production from the murine macrophage cell line RAW 264.7, with a special focus on chemokines. Applying an easy-to-use one-step RT-PCR procedure, we demonstrate the rapid induction of a variety of cytokine/chemokine mRNAs by LPS in the macrophages. In most cases, the message data correlated with ELISA results on the release of the respective mediators. he macrophage plays an important role in immune responses (1). It is highly versatile, very mobile, and responsive to all types of inammatory stimuli. Particularly, monocytes and macrophages are able to release mediators (e.g., cytokines) that activate and modulate other immune and non-immune cells. Studying cytokine release from macrophages may help us understand fundamental mechanisms of inammation. Notably, the macrophage is central not only in host defense but also in wound healing (2). ELISA is used to measure cytokine induction. However, ELISAs require specic antibodies, which are not always available for newly discovered cytokines. Another method to study the induction of a mediator is RT-PCR. Furthermore, mRNA data give additional information when used with ELISA, e.g., when only transcription of a mediator is induced but not translation. We applied one-step RT-PCR as well as ELISA in a simple in vitro system of macrophage cytokine/chemokine induction. The system is the widely used murine cell line RAW 264.7 stimulated with the classical bacteria-derived stimulus lipopolysaccharide (LPS). We show that the 2 methods produce corresponding and complementary results. Importantly, one-step RT-PCR was easy to use and unfailing. METHODS
STIMULATION OF CELLS. Murine RAW 264.7 macrophages (ATCC) were grown in Dulbeccos Modied Eagle Medium (DMEM) with 10% heat-inactivated FBS and penicillin/streptomycin at 37C, 5% CO2 in a humidied atmosphere. Cells were routinely passaged by scraping with a split ratio of 1:5. For ELISA assays, RAW cells were seeded at 2 105 cells/ml in 200 l of medium/well on at-bottomed 96-well microtiter plates in the afternoon the day before stimulation. The next morning, cells were stimulated with 1 g/ml LPS (Salmonella abortus equi) by adding the LPS
Primer (Reference) -actin-s -actin-as IP-10-s (3) IP-10-as (3) KC-s (3) KC-as (3) MIP-2-s (3) MIP-2-as (3) MCP-1-s (3) MCP-1-as (3) MIP-1-s (3) MIP-1-as (3) MIP-1-s (3) MIP-1-as (3) TNF--s (4) TNF--as (4) IL-10-s (5) IL-10-as (5) IL-6-s (4) IL-6-as (4) IL-1-s (4) IL-1-as (4) GM-CSF-s (6) GM-CSF-as (6) Sequence

in 20 l medium/well. The same volume of medium without LPS was added to control wells. After 24 h, supernatants were harvested and stored at 20C, since previous experience had shown that most mediators reached saturation at 24 h. Also, rapidly produced cytokines (e.g., TNF-) accumulated stably in the culture medium. For RT-PCR analysis, stimulation was as described above, but in 1 ml/well on 24-well plates. Based on previous experience, cells were harvested 5 h after stimulation, at a time when macrophages initiate the liberation of most of the mediators measured. Supernatants were discarded and 170 l TRIZOL Reagent were added per well. The cell lysates were harvested after scratching the well with a 200-l pipette tip and multiple resuspensions. Lysates of 6 wells were pooled in a 1.5-ml tube. Total
Product Size (bp) 359 431 530 466 582 561 540 231 300 227 223 435 Tm (C) 56.0 57.0 54.1 58.1 56.5 54.7 54.6 58.3 58.3 59.9 54.9 54.8 54.4 59.7 49.0 42.5 54.6 57.2 51.4 44.3 54.7 49.3 57.7 66.0

ATG GGT CAG AAG GAT TCC TAT GTG CTT CAT GAG GTA GTC AGT CAG GTC CGC ACC TCC ACA TAG CTT ACA G CCT ATC CTG CCC ACG TGT TGA G GAC GAG ACC AGG AGA AAC AGG G AAC GGA GAA AGA AGA CAG ACT GCT TGG GTG GGA TGT AGC TAG TTC C AGT TTG CCT TGA CCC TGA AGC C GGA AAA ATG GAT CCA CAC CTT GC TCT CTT CCT CCA CCA CCA TGC AG GAA GAG TCC CTC GAT GTG GCT A CCC TTT TCT GTT CTG CTG ACA AG CCA CAA TAG CAG AGA AAC AGC AAT AAC CCC GAG CAA CAC CAT GAA G TCT CAT CAG TTC TAT GGC CC GGG AGT AGA CAA GGT ACA AC CTG GAC AAC ATA CTG CTA ACC GAC ATT CAT TCA TGG CCT TGT AGA CAC C GTT CTC TGG GAA ATC GTG GA TGT ACT CCA GGT AGC TAT GG TTG ACG GAC CCC AAA AGA TG AGA AGG TGC TCA TGT CCT CA ATG TGG CTG CAG AAT TTA CTT TTC CT TGG GCT TCC TCA TTT TTG GCC TGG T

TABLE 1. RT-PCR primers. The -actin primers were designed by the author. According to the references, primer sets were designed to amplify a DNA fragment from at least 2 exons of each target so that the fully spliced, mature mRNA product could be distinguished from the unspliced mRNA or contaminating DNA products. s is the sense primer and as is the antisense primer.

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100 bp DNA Ladder

-actin
1 2 1 2 1 2 1 2 Control C 1 2 1 2 1 2 1

MIP-1
2 1 2 C Control 1 2

RNA was isolated as recommended by the manufacturer. RNA was dissolved in 22 l of nuclease-free water and stored at 70C. The quantity of RNA was determined by A260. ONE-STEP RT-PCR. RT-PCR was performed using the S UPER S CRIPT O NE -S TEP RT-PCR System. Briey, one-step RT-PCR was performed on 1 g RNA using 50 pmol each primer (table 1) in a 50-l reaction. Incubation was at 60C for 30 min; 94C for 1 min; then 35 cycles of 94C for 15 s, 60C for 30 s, and 72C for 60 s. A nal extension at 60C for 7 min was performed. After 20, 25, 30, and 35 cycles, 30-s pauses were programmed to obtain 5 l of sample. 2 l loading buffer and 13 l water were added to samples, and 10 l were separated on a 2% agarose gel in 1X TBE with 0.5 mg/L ethidium bromide at 110 V. Bands were visualized with a GIBCO BRL UV Transilluminator, and images and densitometry analysis used the Kodak Digital Science EDAS 120 System. Final images were composed using the Corel 7 software package. ELISA. Antibodies and recombinant cytokines were used at the concentrations recommended by the suppliers. A standard ELISA was performed with NUNC Maxisorp F96 Immunoplates, Certied using an overnight coating, 1 h blocking (10% FBS and 0.05% Tween 20 in PBS; then 2 wash steps with 0.05% Tween 20 in PBS), 4 h sample incubation (then 4 wash steps), 1 h biotinylated antibody (then 4 wash steps), 30 min Streptavidin-POD conjugate (then 5 wash steps), and 10 to 30 min BM Blue incubation. Each plate contained a standard curve (10 ng/ml to 10 pg/ml in duplicate) and 2 blank wells with blocking buffer. The plates were read with a SPECTRAmax Reader, and the data were analyzed using the Softmax Pro 2.2.1. Detection limits for the ELISAs were 10 to
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0 20 25 30 Cycle Number 35

0 20 25 Cycle Number 30 35

FIGURE 1. mRNA induction in RAW 264.7 cells analyzed by one-step RT-PCR. Samples were taken from each tube after 20, 25, 30, and 35 cycles for -actin and MIP-1. Lane 1. Cells not treated with LPS. Lane 2. Cells treated with 1 g/ml LPS. The lanes at 35C are controls done without addition of RT. Lane C. Control without RNA. The graphs are densitometry of the gels.

50 pg/ml, and blank values were below OD 0.1. For samples, ODs between 0.1 and 2.5 were used by analyzing samples in the appropriate dilutions. RESULTS AND DISCUSSION
CYTOKINE/CHEMOKINE MRNA INDUCTION BY LPS.

One-step RT-PCR was used to investigate mRNA induction by LPS. First, as a control, -actin products were analyzed. As expected, the level of -actin products was similar with and without LPS at all cycle numbers (gure 1). The amount of -actin product increased with increasing cycle number and was saturated by 30 cycles. These data indicate that comparable amounts of RNA were subjected to RT-PCR from control and LPS-incubated cells. The importance of sampling at various cycle numbers was more evident

with induction of MIP-1 (macrophage inammatory protein-1). 20 cycles showed induction of message in LPSstimulated cells, but at 25 cycles the unstimulated cells also had a signal, which agrees with the ELISA data below. By 30 cycles, saturation was reached for LPS-stimulated cells, and the difference between control and LPS stimulation was nearly gone. Based on these results (and in accordance with the literature), this method is close to semi-quantitative, as long as cycle numbers are controlled. Furthermore, sampling from one tube during RT-PCR kept RNA consumption and costs low. The ONE-STEP RT-PCR System can be used for semi-quantitative and even quantitative RT-PCR by adding internal controls and adjusting target signals to those,
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100 bp DNA Ladder

or by using kinetic methods to produce quantitative results. However, these methods are more demanding in terms of equipment and experience of personnel compared to the procedures described here, which may hence be more suitable to start with. mRNA induction after LPS stimulation was investigated for selected cytokines/ chemokines. These mediators were induced by the treatment, except for KC (neutrophil-specic chemokine homologous to human GRO-) (gure 2). All PCR products had the predicted sizes. Most of the cytokine messages were constitutively expressed at low levels in unstimulated cells and were highly upregulated by LPS. These experiments show that one-step RT-PCR is highly sensitive, the chosen primers worked under these conditions, and RAW 264.7 macrophages are useful to study induction of a variety of cytokines/ chemokines.
CYTOKINE/CHEMOKINE RELEASE AFTER STIMULATION

600 bp

MIP-2 25

KC 35

MCP-1 25

IP-10 25

MIP-1 25

IL-10 35

TNF- 20

IL-6 35

IL-1 35

GM-CSF 35

FIGURE 2. mRNA induction in RAW 264.7 cells analyzed by one-step RT-PCR. Samples were taken from each tube after 20, 25, 30, and 35 cycles. Products are shown from cycle numbers where reactions behaved linearly (cycle numbers below mediator names). Lane 1. Cells not treated with LPS. Lane 2. Cells treated with 1 g/ml LPS. Controls without RNA or without RT were performed in each case and did not yield products (data not shown).

TNF- Control LPS-Stimulated 0.01 0.01 9.0 1.7 MIP-1 Control LPS-Stimulated 22 2 183 39

IL-6 nd 82 2 MIP-2 1.2 0.4 13 9

IL-10 0.09 0.02 0.07 0.01 KC 0.05 0.01 0.03 0.01

MCP-1 0.8 0.4 55.5 4.6 IP-10 1.7 0.9 60 8

MIP-1 7.6 0.2 103 33 GM-CSF nd 0.88 0.04

TABLE 2. Cytokine/chemokine production in RAW 264.7 cells. Data are mean S.D. in ng/ml for N = 3; nd = not detectable.

Induction of mRNA does not always mean that the protein is produced and released. Mediator release from LPSstimulated macrophages was examined by ELISA. LPS induced the production of most of the mediators analyzed (table 2), with some interesting exceptions. IL-10 was not upregulated by LPS, even though low but signicant mRNA was seen (see gure 2; note the high cycle number). Possibly, IL-10 released at a low level immediately bound in an autocrine manner to receptors on the cells, thus prohibiting detection by ELISA of supernatants. Also, IL-1 was not released from the RAW 264.7 cells (not in the table), despite clear appearance of mRNA. This can be explained by the complex mechanisms of IL-1 release from cells (7). The nascent protein lacks a signal sequence and is produced as a precursor that has to be processed by IL-1 converting enzymes. These enzymes have to be activated
WITH LPS.

themselves, possibly by a second signal. KC was not produced by the macrophages, as seen with RT-PCR. However, induction of KC mRNA and protein was seen in LPSstimulated murine bone marrow-derived macrophages (data not shown). In summary, RAW 264.7 cells are useful to study in vitro cytokine/chemokine release from murine macrophages (except for KC). They can be obtained at high number and purity, and the cells are rather quiescent when not stimulated, unlike the frequently used peritoneal macrophages. Also, the S UPER S CRIPT O NE -S TEP RT-PCR System provides a way for the non-molecular biologist to easily and rapidly obtain mRNA induction data. In most cases, RT-PCR results of mRNA induction corresponded to the ELISA results on protein in the supernatant. Our protocol suggestions may speed up the work of other investigators interested in this area. FOCUS

REFERENCES
1. 2. 3. 4. 5. 6. 7. Gordon, S. (1998) Res. Immunol. 149, 685. DiPietro, L.A. (1995) Shock 4, 233. Su, Y.-H., Yan, X.-T., Oakes, J., and Lausch, R. (1996) J. Virol. 70, 1277. Zhou, H.-R., Yan, D., and Pestka, J.J. (1997) Toxicol. Appl. Pharmacol. 144, 294. Colle, J.-H., Falanga, P.B., Singer, M., Hevin, B., and Milon, G. (1997) J. Immunol. Meth. 210, 175. Ehlers, S., Mielke, M.E., Blankenstein, T., and Hahn, H. (1992) J. Immunol. 149, 3016. Dinarello, C.A. (1998) Int. Rev. Immunol. 16, 457.

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Helpful Tips for Custom Primers

Oligonucleotide Length Percent Full Length 99% Coupling 2 3 4 10 20 30 50 95 99 98.01 97.03 91.35 82.61 74.71 61.11 38.87 98% Coupling 98 96.04 94.12 83.37 68.12 55.66 37.16 14.98

Oligonucleotides are made using computer-controlled DNA synthesizer and the patented parallel array synthesis method. The rst nucleotide is attached to a solid support to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of standard -cyanoethyl chemical reactions. Each cycle consists of: DEBLOCKINGThe rst nucleotide, attached to the solid support via a chemical linker arm, is deprotected by removing the trityl-protecting group. This produces a free 5-hydroxyl group to react with the next nucleotide. COUPLINGThe next nucleotide is added to the reaction and couples (covalently attaches) to the rst nucleotide. CAPPINGAny of the rst nucleotide that failed to react is capped so that it will not play a part in the subsequent synthesis cycles. OXIDATIONThe bond between the rst nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain. DEBLOCKINGThe 5-trityl group is removed from the second nucleotide to prepare it for further cycles. Each cycle results in the addition of a single nucleotide. A chain of nucleotides is built by repeating the synthesis cycle until the desired length is achieved.
What is coupling efciency?

ow are your oligonucleotides made?

Is a small difference in coupling efciency important?

Since coupling efciency is cumulative during DNA synthesis, even a small difference greatly affects the amount of full-length product. Table 1 shows the effect of a 1% difference in coupling efciency and how this inuences the amount of full-length product for different-length oligonucleotides.
Why is trityl group analysis important to perform?

TABLE 1. Coupling efciency affects the amount of full-length primer.

synthesis typically can reach 99%. This means that at every coupling step approximately 1% of the available nucleotides fail to react.
Why is coupling efciency important?

It is used to determine the amount of full-length oligonucleotide produced.

How do I determine the percentage of full-length oligonucleotide?

Every nucleotide added during DNA synthesis has a dimethoxy trityl (trityl) protecting group attached. This trityl group protects the nucleotide from undergoing unwanted chemical reactions during the synthesis cycle and is removed immediately before a new nucleotide is added. We use continuous trityl analysis to measure coupling efciency throughout the synthesis.
How do you measure coupling efciency?

The percentage of full-length oligonucleotide depends on the coupling efciency of the chemical synthesis. The average efciency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula:
% full length= (coupling efciency)n-1, where n = total number of nucleotides. For example, the theoretical yield for a 25-mer would be: 0.9924 = 0.79

The trityl group is colorless when attached to a DNA base but gives a characteristic orange color once removed. The intensity of this color is measured by spectrophotometry and is directly related to the number of trityl molecules present. By comparing the absorbance of trityl throughout synthesis, it is possible to calculate the percentage of nucleotides coupling successfully and, hence, the coupling efciency.
Why do oligonucleotides sometimes require purication?

Coupling efficiency measures how efciently the DNA synthesizer added new nucleotides to the growing DNA chain. If every available nucleotide on the DNA chain reacted successfully with the new nucleotide, the coupling efciency would be 100%. No chemical reaction is 100% efcient. Coupling efciency for DNA
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Therefore, 79% of the oligonucleotide molecules in the tube are 25 bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purication.

Depending on the application and oligonucleotide length, it may be necessary to remove the oligonucleotide that is not full length. Table 2 gives guidelines for the minimum purity for a range of applications.

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Application PCR PCR using primers with critical 5 sequences (e.g., restriction endonuclease sites, RNA polymerase promoters) First-strand cDNA synthesis for RT-PCR Fluorescent sequencing Cycle sequencing Isothermal sequencing Site-directed mutagenesis First-strand cDNA synthesis for generation of libraries GENETRAPPER screening Production of cloning adapters Gel shift assays AFLP analysis CFLP analysis Antisense
TABLE 2. Suggested primer purity.

Minimum Suggested Purity Standard Cartridge Standard Standard Standard Desalted Cartridge Cartridge PAGE Cartridge Cartridge Standard Desalted HPLC purication is cited most frequently

When I order the 50-nmole scale, why dont I receive 50 nmoles of product?

Synthesis scales refer to the amount of starting material present, not the amount of product produced. This is the same for all manufacturers of synthetic DNA. When a 50-nmole scale synthesis is specied, approximately 50 nmoles of the rst nucleotide are added to the DNA synthesizer. Losses occur during processing, transfer of material, and quality control. Oligonucleotide length, sequence, GC content, and coupling efciencies can vary the yield of product. Purication of the oligonucleotide also affects nal product yield as determined by nal OD. On average, the 50-nmole scale will supply enough oligonucleotide for 1,000 to 5,000 PCRs (for 100-l reactions using primer concentration of 0.1 to 0.5 M).
Primer length matters

Calculating primer concentration To calculate the concentration of an oligonucleotide dissolved in 1 ml:

Concentration = A260 Dilution Factor Extinction Coefcient Conversion Factors

The length of the primer affects the yield, as seen in table 3. The table assumes 98% coupling efciency, 100% recovery, and 50% GC content using the 50-nmole scale.
Primer Size (nucleotides) 1020 2030 3040 4050 5060 Expected OD 47 7-9 910 10 10 Expected nmole 4032 3227 2722 2218 1815

Take the A260 reading by diluting 10 l of the oligonucleotide with 990 l of water (1:100 dilution). The A260 value is 0.14 OD. This oligonucleotide has an extinction coefcient of 4.9 nmole/OD.
Concentration = 0.14 OD/ml 100 4.9 nmole/OD 1 mol/103 nmole 103 ml/L = 69 M

To calculate volume to dissolve an oligonucleotide in for a 100-M solution:

Volume = Number of nmoles (1 mol/103 nmole) Desired Concentration Conversion Factor (for L to l)

TABLE 3. Primer length affects yield.

For 24 nmole of an oligonucleotide:

Volume = [24 (1/103)] 106 l 100 mol/L L = 240 l

How do I reconstitute my oligonucleotide?

Dissolve the oligonucleotide in TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. TE is recommended over deionized water since the pH of the water is often slightly acidic and can cause hydrolysis of the oligonucleotide.

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How long can I store the oligonucleotide?

The lyophilized oligonucleotide is stable at 20C for at least 1 year. The oligonucleotide dissolved in TE is stable for at least 6 months at 20C or 4C. The oligonucleotide dissolved in water is stable for at least 6 months at 20C in the absence of nucleases. Be sure the water used is at neutral pH to avoid depurination. Do not store oligonucleotides in water at 4C.
Analysis of oligonucleotides by electrophoresis

Analysis of oligonucleotides by absorbance spectroscopy

Annealing temperature for PCR primers

Unlike large DNA molecules, sequence can affect the migration rate of oligonucleotides. Oligonucleotides of the same size can migrate differently in denaturing polyacrylamide gels, and these differences are composition and sequence dependent. Since migration is sequence dependent, comparison of an oligonucleotide to a molecular size standard or another oligonucleotide of known size is not a reliable way to determine its size. Addition of 40% formamide (in addition to 8 M urea) to the acrylamide gel solution removes most sequence dependent migration effects. [Ausubel, F.M., Brent, R., Kingston, R.E. Moore, D.D., Seidman, J.G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NewYork.] Use of ethidium bromide for detection and quantitation of an oligonucleotide is not reliable. The ability of ethidium bromide to stain oligonucleotides is poor and variable depending on sequence and composition. For more information, see Sewall, A., Natarajan, P., and Fox, D.K. (1999) FOCUS 21, 2.

Properties that are sequence dependent in oligonucleotides include extinction coefcient and A260/280 ratio. For accurate determination of oligonucleotide concentration, calculate the extinction coefcient using an equation that incorporates the contribution of each base and the effect of base stacking [Newton, P.R. (1995) PCR Essential Data, John Wiley and Sons, New York, New York. p.55.] For oligonucleotides, the A260/280 ratio is not an indicator of purity because the ratio of a given oligonucleotide is highly sequence dependent. The reason for this is the large differences in extinction coefcients of individual nucleotides. For more information, see Fox, D. (1998) FOCUS 20, 84.
Guidelines for primer design for PCR

An important parameter for primers is the melting temperature Tm. This is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm is necessary to establish an annealing temperature for PCR. Reasonable annealing temperatures range from 55C to 70C. Annealing temperatures are generally about 5C below the Tm of the primers. Since most formulas provide an estimated Tm value, the annealing temperature is only a starting point. Specicity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures. FOCUS

The ideal PCR primer pair anneals to unique sequences that ank the target and not to other sequences in the sample. Poorly designed primers may amplify other, nontarget sequences. The following guidelines describe the desirable characteristics of a primer sequence: Typical primers are 18 to 24 nucleotides. Select primers that are 40% to 60% GC or mirror the GC content of the template. Avoid complementary sequences at the 3 end of primer pairs. Avoid a GC-rich 3 end. Design primers with G or C residues in the 5 and central regions. Avoid mismatches with the target at the 3 end. Avoid sequences with the potential to form internal secondary structure.

To learn the latest in PCR techniques, enroll in one of our PCR Techniques or Advanced PCR Techniques courses at the Life Technologies Training Center. See inside front cover for the schedule.

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b i o t e c h n o l o g y
Gino Corsini, Augusto Manubens, Manuel Lladser, Sergio Lobos, and Daniela Seelenfreund Department of Biochemistry and Molecular Biology Facultad de Ciencias Qumicas y Farmacuticas Universidad de Chile Casilla 174 correo 22, Santiago, Chile genmolec@abello.dic.uchile.cl Carlos Lobos Programa Nacional de la Mosca de la Fruta Departamento de Proteccin Agrcola Servicio Agrcola y Ganadero (Agriculture and Livestock Service) Bulnes 140, Santiago, Chile

AFLP Analysis of the Fruit Fly Ceratitis capitata

hile Chile was declared free of the med y in 1995, native populations of Ceratitis capitata remain in neighboring countries, so surveillance of over 2,000 miles of border is needed. Identication of the genetic origin of accidentally introduced individuals would help to localize control and facilitate the surveillance. Amplied Fragment Length Polymorphism (AFLP) analysis is an efcient DNA ngerprinting method based on selective amplication using PCR of restriction fragments from a total digest of genomic DNA. The AFLP technique was developed primarily to reveal the differences between cultivars of plant species (1) and has been applied to several crops. AFLP analysis has identied a larger number of molecular markers than RAPDs and RFLP in soybean (2) and cotton (3), among other crops. The AFLP technique has been used to analyze genetic polymorphism of nematodes (45), fungi (6), corals (7), sh (8), and humans (9). One report related to AFLP analysis of arthropod species has been published (10). Polymorphism of wild populations of C. capitata has been studied using multilocus enzyme electrophoresis and RAPDs (1113). In this study, the AFLP technique was used to detect genetic polymorphism in C. capitata (Wiedemann) (Diptera: Tephritidae).

METHODS
MATERIALS. C. capitata from strains Seibersdorf-6096 (S-6), Vienna-60 (a white pupae temperature-sensitive mutant of S-6), Toliman, and Lluta were obtained from Centro de Produccin de Insectos Estriles, C.P.I.E. from the Servicio Agrcola y Ganadero, SAG) located in Lluta, Chile. Toliman is a cross between a native strain from Guatemala (Toliman) and the temperature-sensitive strain Vienna-42 that has been reared at C.P.I.E. since 1996. Lluta 72

strain is a cross of native ies collected in valleys of southern Peru and Azapa (northern Chile) and has been reared at C.P.I.E. since 1993. DNA EXTRACTION . Genomic DNA was prepared from individual larvae or adult specimens. Each individual conserved in 95% ethanol at 20C was dried under vacuum in a 1.5-ml tube and ground at room temperature in 200 l of lysis buffer [50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 3% w/v SDS, 0.1 M 2-mercaptoethanol] with 25 l proteinase K (10 mg/ml) until the solution turned a reddish color. Another 200 l of lysis buffer were added, and the mixture incubated for 10 min at 65C. 100 l of 5 M potassium acetate were added, and the mixture was incubated on ice for 10 min. After centrifugation for 10 min at 12,000 g, the supernatant was extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). 0.6 volumes of cold isopropanol was added to the supernatant and incubated at 20C for 20 min. The DNA was collected by centrifugation, and the pellet was washed with 70% ethanol, dried, and dissolved in 50 l TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)]. DNA was incubated with 10 l RNase A (10 mg/ml) for 45 min at 37C and then treated with 10 l of proteinase K (10 mg/ml) for 15 min at 50C. TE was added to 400 l, and the sample was extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). The DNA precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of cold ethanol. After centrifugation, the DNA pellet was washed in 70% ethanol, dried, and dissolved in 40 l of TE buffer. AFLP REACTIONS. AFLP products were generated using the GIBCO BRL AFLP Analysis System I according to the manufacturers instructions. GIBCO BRL Taq DNA Polymerase was used for PCR. The

AFLP products (5 l) were separated on 5.8% or 6.0% (w/v) acrylamide gels at 70 to 80 W (1,700 1,800 V) in 1X TBE on a Model S2001 gel electrophoresis system. After electrophoresis, the gel was dried and exposed to a Kodak X-Omat AR lm.
ISOLATION AND CLONING OF POLYMORPHIC GENETIC

The selected bands were cut directly from the dry gel, submerged in TE buffer containing 1 M NaCl, and incubated overnight at 37C. The solution was collected and ethanol precipitated at 20C for 30 min. After centrifugation at 12,000 g for 20 min, the DNA was vacuum dried and dissolved in 20 l of sterile double-distilled water. The sample was amplied using the same pair of primers selected from the AFLP reaction. PCR was 35 cycles of 94C for 30 s, 56C for 30 s, 72C for 1 min, and a nal soak at 4C. The amplied DNA was visualized on a 3% agarose gel stained with ethidium bromide. The DNA was ethanol precipitated, and the pellet was washed with 70% ethanol, dried, and dissolved in sterile water. The DNA was ligated to a vector and transformed into DH5F competent cells. Both strands of the clones were sequenced with the GIBCO BRL dsDNA Cycle Sequencing System. Sequences were analyzed using the BLAST program from NCBI.
MARKERS.
1 2 3 4 M 5 6

FIGURE 1. Puried genomic DNA. DNA was extracted from individual adult ies or larvae from different strains, and 1/4 of the samples were electrophoresed on 0.8% agarose TBE gels. Lanes 13. Strain Lluta (male, female, and larva, respectively). Lane 4. Strain S-6 (larva). Lanes 5 and 6. Strain Toliman (males), digested with EcoR I (lane 5) or undigested (lane 6). Lane M. DNA/Hind III fragments.

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25 bp DNA Ladder

25 bp DNA Ladder

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1 2 & ( & ( & (&(

RESULTS AND DISCUSSION Puried genomic DNA from an adult y or larvae yielded 4 to 5 g of unsheared DNA with a 260/280 ratio of 1.6 to 1.8 (gure 1). In initial AFLP experiments, 2 similar strains were screened with 64 primer combinations for at least one differentiating band (data not shown). Generally, primer pairs produced 35 to 50 bands from 75 to 500 bp, but few primer pairs produced distinctive patterns. Based on the initial screening, 8 primer pairs were chosen to analyze both sexes of 4 laboratory strains. The primer pair E-AAG/M-CAT identied specic markers of Lluta, Toliman, and Vienna-60 in both male and female individuals (gure 2). S-6 presented a characteristic marker with E-AGG/M-CAG primers (data not shown). The AFLP patterns from several DNA samples of the same individual were reproducible (data not shown). However, ies belonging to the same strain exhibited a number of bands that varied between individuals. Therefore, our analysis was focused on identifying marker bands that were common for all individuals of a specic strain. For example, comparing individuals from the Lluta strain, the expected common marker band was seen with both primer pairs, as well as a similarity in the banding pattern (gure 3). However, a number of bands reecting individual differences (and high genetic diversity) were observed, even though this strain has been reared in the laboratory for >30 generations. These assays were performed for the other strains, and the presence of the respective marker bands was detected for Toliman and Vienna-60 (data not shown). To use the marker bands as specic probes, we have isolated and cloned the genetic marker bands from Vienna-60, Lluta, and Toliman. The fragments were 280 bp (Lluta), 360 bp (Vienna 60), and
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FIGURE 2. Comparison of AFLP patterns. Strains were analyzed using the selective primer pair E-AAG/M-CAT. Lanes 1 and 2 correspond to the analysis of larval tissue from strains S-6 and Lluta, respectively. All other lanes show DNA extracted from adult ies. Lane 3. Strain Lluta. Lane 4. Strain S-6. Lane 5. Strain Toliman. Lane 6. Strain Vienna 60. For each strain, AFLP patterns from one male (() and one female (&) are shown. Arrows indicate characteristic marker bands for each strain using this primer pair.

FIGURE 3. Presence of marker band in strain Lluta individuals. 5 males (lanes 15) and 5 females (lanes 610) were analyzed with E-AAG/M-CAT (panel A) and E-AAC/M-CAA (panel B). Arrows indicate the presence of the characteristic marker band.

210 bp (Toliman). All fragments were A/T rich (data not shown). A search of GENBANK for these fragments did not match with sequenced genes from Drosophila or Ceratitis. PCR and Southern hybridization are in progress to verify that these marker bands can be used as specic probes. In summary, we have applied the AFLP technique to identify laboratory strains of med ies. Our goal is to map the genetic diversity of native wild med y populations from different geographical locations surrounding the Chilean borders. Preliminary analysis of wild populations from different geographical locations suggests that local patterns can be obtained. The feasibility of isolating specic markers for each strain could eventually facilitate the analysis and monitoring of y populations.

ACKNOWLEDGEMENTS This work was nanced by the joint project FAC-SAG/PA-001/97 from the Facultad de Ciencias Qumicas y Farmacuticas, Universidad de Chile, and the Servicio Agrcola y Ganadero. FOCUS REFERENCES
1. 2. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van der Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. (1995) Nucl. Acid Res. 23, 4407. Lin, J.J., Kuo, J., Ma, J., Saunders, J.A., Beard, H.S., MacDonald, M.H., Kenworthy, W., Ude, G.N., and Matthews, B.F. (1996) Plant Mol. Biol. Rep. 14, 156. Feng, X., Saha, S., and Soliman, K. (1997) Focus 19, 11. Roos, M.H., Hoekstra, R., Plas, M.E., Otsen, M., and Lenstra, J.A. (1998) J. Helminthol. 72, 291. Semblat, J.P., Wajnberg, E., Dalmasso, A., Abad, P., and Castagnone-Sereno, P. (1998) Mol. Ecol. 7, 119. Mueller, V.G., Lipari, S.E., and Milgroom, M.G. (1996) Mol. Ecol. 5, 119. Lopez, J.V., Kersanach, R., Rehner, S.A., and Knowlton, N. (1999) Biol. Bull. 196, 80. Liu, Z., Nichols, A., Li, P., and Dunham, R.A. (1998) Mol. Gen. Genet. 258, 260. Schreiner, T., Prochnow-Calzia, H., Maccari, B., Erne, E., Kinzler, I., Wolpl, A., and Wiesneth, M. (1996) J. Immunol. Methods 196, 93. Reineke, A., Karlovsky, P., and Zebitz, C.P. (1998) Insect Mol. Biol. 7, 95. Baruf, L., Damiani, G., Gugliemino, C.R., Bandi, C., Malacrida, A.R., and Gasperi, G. (1995) Heredity 74, 425. Haymer, D.S. and McInnis, D.O. (1994) Genome 37, 244. Sonvico, A., Manso, F., and Quesada-Allue, L.A. (1996) J. Econ. Entomol. 89, 1208.

3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13.

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Joe Crouse, Teresa Myers, and Julie Brent Technical Services Part of the Technical Support and Training Team Life Technologies, Inc. Rockville, Maryland 20849

Buffer Compatibility for Common Reactions

aving time and reagents are objectives shared by many molecular biologists. If 2 enzymes can be used in the same reaction buffer, it could mean using both enzymes at the same time or at the very least it could save the time and resources associated with purication between the reactions. This piece addresses many commonly asked questions related to buffer compatibility.

REACT buffers 1, 2, 3, 4, 6, or 10. Additionally, it describes a protocol where calf intestinal alkaline phosphatase can be added directly at the beginning of a restriction digest using the REACT buffers mentioned above. The reaction must be incubated for at least 1 h. The bulletin can be found in the TECH-ONLINE section of our web site in the Molecular Biology manuals and technical bulletin section.
Can T4 DNA ligase be used in any REACT buffer?

Can you use a PCR buffer with DNase I?

How do I choose a REACT buffer for my double digest with restriction endonucleases?

GIBCO BRL Restriction Endonucleases are provided with a REACT Buffer to ensure optimal performance. We do not recommend double digests because of possible buffer incompatibility problems and enzyme steric hindrance problems. However, we know many researchers perform double digests to save time. Choose the combination of buffers that yields the highest activity for both enzymes. It is possible to use a REACT buffer that gives less than 100% enzyme activity for a particular restriction endonuclease and increase the amount of enzyme used in the reaction. Enzyme buffer activity charts can be found on pages R-52 and R-53 in the 20002001 GIBCO BRL Catalogue, or search for double digests in the TECH-ONLINE section of our web site.

SM

No. T4 DNA ligase comes with a 5X T4 DNA ligase buffer [250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% polyethylene glycol]. None of the REACT buffers are comparable to the ligase buffer. The REACT buffers lack the ATP necessary for the ligase reaction. The 5X T4 DNA Ligase Buffer is available separately.
What is the activity of restriction endonucleases in PCR buffer?

Yes. DNase I does not come with a buffer, so the Taq 10X PCR buffer is an alternative. Amplication Grade DNase I does come with a 10X reaction buffer [200 mM Tris-HCl (pH 8.3), 500 mM KCl, and 20 mM MgCl2]. The Taq 10X PCR buffer and the Amplication Grade DNase I buffer have the same formulation. The buffers of the specialized PCR enzymes like E LONG ASE Enzyme Mix and PLATINUM Pfx DNA Polymerase have not been tested with DNase I.
Since most primers are purchased without 5 phosphates, can T4 polynucleotide kinase be added to PCR?

Is there a simplied protocol for using alkaline phosphatase that does not require phenol extraction and ethanol precipitation after restriction endonuclease digestion?

Yes. In fact, Technical Bulletin 18009-1 describes the activity of calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, and temperature-sensitive alkaline phosphatase in 6 common restriction endonuclease buffers. If enough of each enzyme (1 unit, 150 units, and 1 unit, respectively) is used, 100% enzyme activity is seen in
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Although common PCR buffer [TrisHCl (pH 8.4), MgCl2, and KCl) would support restriction endonuclease activity, we do not suggest adding restriction endonucleases directly to PCR products. Even though restriction endonuclease reactions are done at lower temperatures than Taq DNA polymerase reactions, Taq DNA polymerase is still active at these temperatures. This means that 5 overhangs generated by restriction endonucleases will be lled in by the Taq DNA polymerase. Therefore, the best approach would be to clean up the PCR using a product such as the CONCERT Rapid PCR Purication System prior to the digestion [see FOCUS (1999) 21, 11]. Alternatively, the use of TAQUENCH PCR Cloning Enhancer will inhibit the Taq DNA polymerase activity during the restriction digestion [see FOCUS (1998) 20, 15].

At best, T4 polynucleotide kinase would show partial activity. The pH of most PCR buffers is slightly above the optimum pH range of 7.4 to 8.0 for the forward kinase reaction. The MgCl2 is lower than the suggested optimum of 10 mM. Additionally, the accumulated pyrophosphate from the PCR would further reduce the kinase activity. The remaining dNTPs from the PCR could, however, serve as a substrate for the kinase. If the objective, however, is to get signicant phosphorylation, this approach is strongly discouraged.
Can T4 DNA polymerase be added to the second-strand cDNA synthesis reaction to polish the cDNA ends?

Yes. In fact, in all of the SUPERSCRIPT Systems for cDNA Synthesis, 10 units of T4 DNA polymerase are added directly to the second-strand synthesis reaction. The composition of second-strand synthesis reaction is 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)SO4, 0.15 mM -NAD+, and 1.2 mM DTT.

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Are any of my REACT buffers compatible with the Klenow (Large Fragment DNA Polymerase I)?

Klenow buffer is in essence REACT 2 buffer 10X [500 mM Tris-HCl (pH 8.0), 100 MgCl2, and 500 mM NaCl]. Before polishing the ends of a cut fragment, heat inactivate the restriction endonuclease digestion for 10 to 15 min at 65C. For enzymes that are not heat sensitive, do a phenol-chloroform extraction. To nd out how active your restriction endonuclease is in REACT 2 buffer, check the reference section in the Life Technologies 20002001 Catalogue (page R-52).
What is the best buffer to use to avoid an ethanol precipitation step between a ligation and kinase reaction?

Oftentimes, when cloning adapters are added to fragments to be subcloned, only one end is phosphorylated. This means that once the fragment is ligated to the adapters, 5 phosphates will need to be added to the resulting product. T4 polynucleotide kinase will show some activity in PEG-containing T4 DNA ligase buffers. In the SUPERSCRIPT Choice System for cDNA Synthesis, however, the use of a unique 5X Adapter Buffer [330 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP] is recommended for the adapter ligation reaction and the phosphorylation reaction. The ligation of the adapters is done in 1X buffer at 16C. After heat inactivating the T4 DNA ligase at 70C for 10 min, T4 polynucleotide kinase is added to this reaction for the kinase step. FOCUS

I study the biomolecular mechanisms of vitamin C deciency diseases. I like to think of myself as a Ricket Scientist.

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How Basal Media Provide theCulture Optimal Environment for Cell

o maximize success, the in vitro culture conditions are set up to mimic in vivo conditions of temperature, pH, osmolality, and nutrition. Cell culture medium maintains pH and osmolality essential for viability and provides the nutrients and energy required for cell growth. Basal cell culture media minimally consist of salts, amino acids, vitamins, and energy sources. This paper discusses the functions of these components of basal media.

Kevin Grady Technical Services Part of the Technical Support and Training Team Life Technologies, Inc. Grand Island, New York 14072

PH AND OSMOTIC PRESSURE

The salts in basal medium are primarily responsible for providing physiological pH and osmotic pressure, membrane potential, enzyme cofactors, and cell attachment. Culture media are buffered to compensate for the cellular production of CO2 and lactic acid as metabolic by-products. Traditionally, basal cell culture media have been buffered by bicarbonate (HCO3). As cells grow, CO2 is evolved. The dissolved CO2 forms a buffering system with the bicarbonate. However, if cell density is low or cells have entered into a lag phase, they may not produce sufcient CO2 to maintain optimal pH. To counter these potential problems, bicarbonate-buffered media require the use of incubators with an atmosphere of 5% to 10% CO2. Media with low levels of bicarbonate, such as MEM (1.5 to 2.2 g/L), require 5% CO2; media like D-MEM (3.7 g/L), with higher levels of bicarbonate, require 10% CO2 to maintain correct pH. The important factor is using the correct percent CO2 based upon a mediums bicarbonate level to maintain physiological pH, irrespective of cell type.

ENERGY SOURCES Carbohydrates, primarily D-glucose, and the amino acid L-glutamine are the major carbon and energy sources in media. Cultured cells have two primary paths of energy (ATP) production: conversion of glucose to lactate (or full oxidation to CO2) and oxidation of L-glutamine to CO2 and ammonia. Traditional glucose levels in culture media range from 1 to 4.5 g/L. Generally, the metabolic rate of a cell line correlates to the optimal glucose level. A slow-growing cell line (long doubling time) will grow in low or high glucose. However, a fast-growing cell line requires higher glucose levels to maintain its metabolic rate. Exposure to lower-than-optimal glucose levels could induce the cells to enter a lag phase. A signicant portion of the energy required to maintain cell growth (30% of the energy produced; this can vary with cell type) comes from the oxidation of L-glutamine. Typical ranges for L-glutamine in formulations are 1 to 4 mM, 10 times the concentration of other amino acids, due to L-glutamines importance as an energy source as well as its role in protein synthesis. The high L-glutamine concentration present in formulations may also be due to its somewhat unstable nature in solution. To counter this, a dipeptide form of L-glutamine (GLUTAMAX I, L-alanyl-L-glutamine; GLUTAMAX II, glycyl-L-glutamine) can be substituted for L-glutamine in medium. These dipeptides are extremely stable in solution and can be autoclaved without decomposition. Another energy/carbon source that is present in media is pyruvate. In carbohydrate catabolism (glycolysis), D-glucose is converted via several reactions to pyruvate. Pyruvate can be oxidized further to produce energy and CO2 or converted to lactate.

NUTRIENTS Amino acids are incorporated into proteins. At a minimum, basal medium must contain the essential amino acids those amino acids that cannot be synthesized at a rate adequate to meet metabolic requirements. More specialized medium formulations, developed for low serum supplementation or high cell density, often have non-essential amino acids added to ensure that amino acids do not limit the maximum cell concentration attainable. Vitamins are needed for cell growth and multiplication. Vitamins are precursors of enzymatic cofactors. Vitamin concentration is important in terms of cell survival and growth rate rather than directly affecting maximum cell density. For most applications, basal media generally require serum supplementation. Serum is largely undened, but it supplies essential growth factors and hormones required for cell growth. Serum also provides an additional source of carbohydrates, amino acids, and vitamins. Therefore, serum supplementation lessens the concerns about a basal medium choice and whether a specic formulation provides the essential levels of carbohydrates, amino acids, and vitamins. The growth-promoting factors present in serum provide a mechanism to easily adapt cells from one formulation to another. In a serum-supplemented application, choice of basal medium may be secondary to using the right serum. FOCUS

To learn more about cell culture, enroll in our Cell Culture Techniques or Advanced Cell Culture Techniques courses at the Life Technologies Training Center. See inside front cover for the schedule.

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