Professional Documents
Culture Documents
French
BioBrickMasterClass21Jul10.doc
iGEM UK Meeting
21 July 2010
http://partsregistry.org/Main_Page
http://2009.igem.org/Partner_Offers
Template
C. French
http://www.dsmz.de/
http://www.ncimb.com/
http://www.lgcstandards-atcc.org/
Primers
• make sure you get the prefix and suffix right
• assembly standard 10 is recommended, or 23 if fusion proteins are needed.
• if you want to use a different assembly standard, you must obtain a variance
from iGEM HQ. Apply as early as possible.
http://partsregistry.org/Help:Contents
http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix
http://www.biobricks.org/
Polymerase
• Taq is fast and cheap and leaves A-overhangs, but has a strong tendency to
introduce mutations.
• Pfu is much more accurate but slower and more expensive.
• New polymerase such as Kod, Velocity and Phusion are fast and accurate.
Reaction conditions
• Templates with strong secondary structure can be amplified by increasing the
denaturation time to 1 minute per cycle and including 10% v/v glycerol in the
reaction.
5. Site-directed mutagenesis
http://www.openwetware.org/wiki/French_Lab
6. Sequence checking
7. Registry Vectors
http://partsregistry.org/Plasmid_backbones
http://partsregistry.org/Plasmid_backbones/Assembly
Important note
Ligations give you a complicated mixture of products. The problem in assembly is to
select the right one. This can be done either by cloning or PCR.
Standard method
• excise the upstream part with E/S, cut the downstream construct with E/X, and
ligate.
• Or, excise the downstream part with X/P, cut the upstream part with S/P, and
ligate.
• Problems: often low efficiency, no way to select: many minipreps!
http://partsregistry.org/Assembly:Standard_assembly
Three-antibiotic method
• Excise upstream part with E/S, excise downstream part with X/P, and ligate
into a vector cut with E/P which carries a different antibiotic resistance marker
to those of both the upstream and downstream parts.
• Perform triple ligation.
• May require vector exchange.
C. French
http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly
BALTIC
• An assembly method using an extra restriction site in the vector outside the
BioBrick region, usually ScaI.
• High efficiency.
• Requires ScaI sites to be absent from both parts.
• See my OpenWetware site for details.
• superceded now, but might still be interesting if you are having problems with
a particular construct.
http://www.openwetware.org/wiki/French_Lab
uuuuuuuuuuuuuuuuuuuu-a ctaga-ddddddddddddddddd
uuuuuuuuuuuuuuuuuuuu-tgatc t-ddddddddddddddddd
http://ginkgobioworks.com/