Difference between revisions of "Template:SBB-Protocols LRGtw"

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(LR Gateway Transfers)
 
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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.
 
===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'''
 
'''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