[[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. <br><br> ---++++_Assembly Reaction_ *1.* Set up the following reaction mix: <div style="padding: 1%"> * 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* </div> *2.* Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions: <div style="padding: 2%"> | *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 | </div> *3.* Transform 2 μL assembly reaction into [[ProtocolsElectrocompetentCells][Electrocompetent]] or [[ProtocolsChemCompCellsHub][Chemically Competent]] cells and plate on LB + Cam <br><br> ---++++_Expected Results_ * (If using pYTK001 as your entry vector) Multiple non-fluorescent colonies * Be sure to screen colonies for GFP expression on a blue light transilluminator, pick colonies that do not fluoresce! * Cells containing properly assembled plasmids (confirmed through PCR and Sanger Sequencing) _If you are having repeated issues check out the [[ProtocolsGoldenGateTroubleshooting][Troubleshooting]] page!_ --- [[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]]
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Topic revision: r10 - 2021-07-09 - KateElston