[[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]] ---+ Part Plasmid Assembly Once you have designed your part and either amplified with PCR or ordered the desired gBlock (as described [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKDesignANewPart 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 | 42°C | 1.5 min | | 2 | 16°C | 3 min | | Cycles 1-2: | Repeat 25x | | | 3 | 50°C | 5 min | | 4 | 80°C | 10 min | * Transform 2 μL assembly reaction and plate on LB + Cam * Screen colonies for gfp dropout using blue light box, pick colonies that do not fluoresce --- [[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]] --- -- Main.KateElston - 29 Jan 2018
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ProtocolsBTKMakeANewPartPlasmid
Topic revision: r9 - 2021-06-29 - VictorLi