Template:SBB-Protocols LRGtw: Difference between revisions
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===LR Gateway Transfers=== | |||
In preparation for doing the Gateway reaction, check out the following page:<br> | In preparation for doing the Gateway reaction, check out the following page:<br> | ||
http://2008.igem.org/Team:UC_Berkeley/GatewayOverview | http://2008.igem.org/Team:UC_Berkeley/GatewayOverview | ||
You will be doing a normal ''in vitro'' LR gateway reaction. The pBca1254** vectors all contain the attR1 and attR2 which will recombine with the attL1 and attL2 sites in your donor pBca1256 plasmids. Upon reaction, the lethal ccdB gene in the pBca1254** assembly vector will be displaced allowing for selection of your product after transformation. The name of your product plasmid will be pBca9495**-partname. | You will be doing a normal ''in vitro'' LR gateway reaction. The pBca1254** vectors all contain the attR1 and attR2 which will recombine with the attL1 and attL2 sites in your donor pBca1256 plasmids. Upon reaction, the lethal ccdB gene in the pBca1254** assembly vector will be displaced allowing for selection of your product after transformation. The name of your product plasmid will be pBca9495**-partname. | ||
'''There are no restriction enzymes, ligases, gels, or zymo columns in the Gateway reaction! You just mix plasmids and enzyme, let it cook, then transform''' | '''There are no restriction enzymes, ligases, gels, or zymo columns in the Gateway reaction! You just mix plasmids and enzyme, let it cook, then transform''' | ||
*Set up the following mixture in a 0.5mL microcentrifuge tube | *Set up the following mixture in a 0.5mL microcentrifuge tube | ||
Latest revision as of 15:05, 22 January 2014
LR Gateway Transfers
In preparation for doing the Gateway reaction, check out the following page:
http://2008.igem.org/Team:UC_Berkeley/GatewayOverview
You will be doing a normal in vitro LR gateway reaction. The pBca1254** vectors all contain the attR1 and attR2 which will recombine with the attL1 and attL2 sites in your donor pBca1256 plasmids. Upon reaction, the lethal ccdB gene in the pBca1254** assembly vector will be displaced allowing for selection of your product after transformation. The name of your product plasmid will be pBca9495**-partname. There are no restriction enzymes, ligases, gels, or zymo columns in the Gateway reaction! You just mix plasmids and enzyme, let it cook, then transform
- Set up the following mixture in a 0.5mL microcentrifuge tube
3uL ddH2O 0.5uL Donor plasmid (a pBca1256-*) 0.5uL Recipient plasmid (a pBca1254**-*)
- Add 1uL of LR Clonase
- Slam on bench upside down to mix
- Quick spin to send it to the bottom of the tube
- Incubate at 25 degrees on the thermocycler for 1hr
- Add 0.5uL proteinase K
- Slam on bench upside down to mix
- Quick spin to send it to the bottom of the tube
- Incubate at 37 degrees on the thermocycler for 10min.
- Put on ice, proceed to transformation
