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Second Stage Assembly

Second Stage assemblies are used to build plasmids out of multiple transcriptional units (Protocol found here). Occasionally this assembly can be used as a second step to building troublesome first stage plasmids - the first stage plasmid is assembled in a high copy/easier to work with backbone then moved to lower copy/more difficult to work with backbone. This assembly makes use of the BsmBI enzyme as described below.

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each transcriptional unit plasmid for this assembly. Various backbones are regularly used in this step - if toxicity issues may be a factor, lower copy number vectors are recommended.

  • 10 fmol of each transcriptional unit/backbone
  • 2 μL of 10 T4 DNA ligase buffer (Promega)
  • 1 μL of BsmBI
  • 1 μL of T4 DNA ligase
  • x μL water up to 20 μL total.

Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions:

Step Temperature Time
1 42C 1.5 min
2 16C 3 min
Cycles 1-2: Repeat 25x  
3 50C 5 min
4 80C 10 min

  • Transform 2 μL assembly reaction and plate recovery on LB + Selective Antibiotic

Check out troubleshooting tips here


-- Main.KateElston - 29 Jan 2018

 Barrick Lab  >  BroadHostRangeToolkit  >  ProtocolsBTKAssembleMultipleTUPlasmid

Topic revision: r4 - 22 Mar 2018 - 19:26:14 - Main.KateElston
 
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