(r10) ProtocolsBTKMakeANewPartPlasmid < Lab

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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 [[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.
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---++++_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 1-2&mu;L]
   * 20 fmol of insert DNA = 650 * insert length * 20x10^-6 = *X ng* [try to keep volume to less than 10 &mu;L]
   * 2 &mu;L of 10× T4 DNA ligase buffer (Promega)
   * 1 &mu;L of BsmBI
   * 1 &mu;L of T4 DNA ligase
   * x &mu;L water up to *20 &mu;L 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 &mu;L assembly reaction into [[ProtocolsElectrocompetentCells][Electrocompetent]] or [[ProtocolsChemCompCellsHub][Chemically Competent]] cells and plate on LB + Cam
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---++++_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!_

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Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKMakeANewPartPlasmid
Topic revision: r10 - 2021-07-09 - 22:28:09 - Main.KateElston