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.

Protocol source: NEB ([[https://www.neb.com/protocols/2020/01/15/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-kit-bsmbi-v2-neb-e1602]])
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---++++_Assembly Reaction_
[[Old_Golden_Gate_Assembly][Access the old non-kit golden gate assembly protocols here]]

*1.* Calculate the mass (in ng) required for 50 fmol of vector and 100 fmol of insert using NEB's NEBioCalculator: [[https://nebiocalculator.neb.com/#!/dsdnaamt]]

*2.* Set up the following reaction mix: 
<div style="padding: 1%">
   * 50 fmol pYTK-001 plasmid = *82.68 ng*  [try to keep volume to 1-2&mu;L]
   * 100 fmol of insert DNA = 650 * insert length * 100x10^-6 = *X ng* [try to keep volume to less than 10 &mu;L]
   * 2 &mu;L of NEB 10× T4 DNA ligase buffer 
   * 1 &mu;L of NEB Golden Gate Enzyme Mix (BsmBI-v2)
   * x &mu;L water up to *20 &mu;L total*
</div>
*3.* 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>

Optionally, you may have high enough efficiency if inserting one PCR product into the entry vector with NEB's short protocol with no cycling instead of the longer protocol:
<div style="padding: 2%">
|  *Step*  |  *Temperature*  |  *Time*  |
|  1  |  42°C  |  5 min  |
|  2  |  60°C  |  5 min |
</div>

*4.* 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_
<img src="%ATTACHURLPATH%/Part_Plasmid_Assembly.png" alt="Part_Plasmid_Assembly.png" width="670" height="281" />

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   * (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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This topic: Lab > BroadHostRangeToolkit > ProtocolsBTKMakeANewPartPlasmid
Topic revision: r16 - 2022-06-30 - JeffreyBarrick