(r9) Barrick Lab :: ProtocolsBTKMakeANewPartPlasmid

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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.

---+++ Assembly reaction

Total volume will be 20 &mu;L; 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 1-2&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.
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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 &mu;L assembly reaction and plate on LB + Cam
   * Screen colonies for gfp dropout using blue light box, pick colonies that do not fluoresce

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 -- Main.KateElston - 29 Jan 2018

Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKMakeANewPartPlasmid

Contributors to this topic

KateElston, JeffreyBarrick, DennisMishler, VictorLi, PatrickLariviere, SeanLeonard

Topic revision: r9 - 2021-06-29 - 20:04:09 - Main.VictorLi