Difference: ProtocolsBTKMakeANewPartPlasmid (12 vs. 13)

Revision 132021-11-03 - PatrickLariviere

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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 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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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)

Assembly Reaction

Access the old non-kit golden gate assembly protocols here

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1. Set up the following reaction mix:

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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

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2. Set up the following reaction mix:

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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

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50 fmol pYTK-001 plasmid = 82.68 ng [try to keep volume to 1-2μL]
100 fmol of insert DNA = 650 * insert length * 20x10^-6 = X ng [try to keep volume to less than 10 μL]
2 μL of NEB 10× T4 DNA ligase buffer
1 μL of NEB Golden Gate Enzyme Mix (BsmBI-v2)

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1 μL of T4 DNA ligase

x μL water up to 20 μL total

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2. 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

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3. Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions:

42°C for 5 min
60°C for 5 min

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2	16°C	3 min
Cycles 1-2:	Repeat 25x
3	50°C	5 min
4	80°C	10 min

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4. Transform 2 μL assembly reaction into Electrocompetent or Chemically Competent cells and plate on LB + Cam

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3. Transform 2 μL assembly reaction into Electrocompetent or Chemically Competent cells and plate on LB + Cam

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 Troubleshooting page!

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