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Part Plasmid Assembly

Once you have designed your part and either amplified with PCR or ordered the desired gBlock (as described here) you can proceed to the assembly step of the part plasmid itself. The example reaction below shows pYTK001 used as the entry vector for the reaction; however, this can be substituted for any other entry vector with requisite BsmBI cut sites. Once built, part plasmids can be assembled into transcriptional units.

Assembly reaction

Total volume will be 20 μL; You will need 10 fmol of entry vector and 20 fmol of your DNA insert(s).

  • 10 fmol pYTK-001 plasmid = 17.7ng [try to keep volume to 1-2μL]
  • 20 fmol of insert DNA = 650 * insert length * 20x10^-6 = X ng [try to keep volume to less than 10 μL]
  • 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 on LB + Cam

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-- Main.KateElston - 29 Jan 2018

 Barrick Lab  >  BroadHostRangeToolkit  >  ProtocolsBTKMakeANewPartPlasmid

Topic revision: r7 - 14 Aug 2018 - 19:56:57 - Main.KateElston
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