Amplifying a Plasmid

Sandra March 31, 2004

If you receive a plasmid from a collaborator in the mail, you will usually get only a few microlitres.
It's better not to amplify the plasmid in protein expression strains, since they are not always well
suited to plasmid extraction and purification. To amplify, transform into E. coli DH5α , pick a
single colony and do a mini-plasmid preparation as usual. Check with the donator or the
appropriate reference what is the antibiotic selection for the plasmid. It's usually ampicillin, but
can also kanamycin, tetracycline or chloramphenicol. Second, find out the copy number of the
plasmid (low or high). This will determine how much culture is grown for plasmid extraction.

Transformation of Subcloning Efficiency DH5α

• Obtained from Biochemistry Stores, Invitrogen supply center. Cat #18265-017.

• Comes in 4 X 0.5 ml aliquots. Stored at -80°C. Bring dry ice when you buy the cells.
• The cells need to be aliquoted. Follow the instructions provided in the manual.
• Thaw a vial on wet ice.
• Promptly aliquot in 50 µ l aliquots and re-freeze in a dry ice/ 95% ethanol bath.
Mix the cells after thawing, by gentle inversion.
Prechill the tubes before adding cells.

1. Thaw an aliquot of cells on wet ice.

2. Add 0.5 to 1 µ l of plasmid. If plasmid concentration is given, dilute to 10 ng/µ l and use 1
µ l . Mix by tapping the tube gently. Leave on ice for 30 min.
3. Heat-shock for exactly 20 seconds in a 37°C water bath.
4. Place back on ice for 2 min.
5. Add 1 ml LB or SOC medium. Place the tube on its side in a beaker and shake at 37°C at 225
rpm for 1 hour.
6. Spread 100 µ l on an LB agar plate containing the appropriate antibiotic (see below*). If it's
ampicillin, use plates containing 100 µ g/ml carbenicillin (LB+CB). If the plasmid
concentration was not given, spread 10 µ l of the cells on a second plate, containing a 100 µ l
pool of LB media. Store the remaining cells in at 4°C.
7. Incubate at 37°C overnight (about 16 hours) to allow colonies to form. If the colonies are too
dense, plate 1 µ l cells in a 100 µ l pool of LB on a fresh LB+CB plate.
8. Inoculate 4-10 ml of LB containing the appropriate antibiotic with a single colony. Grow
overnight (16-18 hours) at 37°C, 240 rpm.
9. Isolate plasmid as usual (see Mini plasmid preparation). The volume of culture used depends
on the plasmid copy number. See the Qiagen plasmid prep manual for a discussion of
plasmid copy number. Make a glycerol stock of the bacteria and place in the lab plasmids and
bacterial strains box. Remember to update the spreadsheet on the computer.

*Antibiotics:
KanR: 50 µ g/ml kanamycin
AmpR: 100 µ g/ml carbenicillin/ampicillin
TetR: 12.5 µ g/ml tetracycline
CamR: 25 to 34 µ g/ml chloramphenicol