Template:SBB-Protocols LRGtw

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.

LR Gateway Transfers

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