Second Stage Assembly
Second Stage assemblies are used to build plasmids out of multiple transcriptional units (
Protocol found here). Occasionally this assembly can be used as a second step to building troublesome first stage plasmids - the first stage plasmid is assembled in a high copy/easier to work with backbone then moved to lower copy/more difficult to work with backbone. This assembly makes use of the BsmBI enzyme as described below.
Assembly reaction
Total volume will be 20 μL; you will need 10 fmol of each transcriptional unit plasmid for this assembly. Various backbones are regularly used in this step - if toxicity issues may be a factor, lower copy number vectors are recommended.
- 10 fmol of each transcriptional unit/backbone
- 2 μL of 10× T4 DNA ligase buffer
- 1 μL of BsmBI
- 1 μL of T4 DNA ligase
- x μL water up to 20 μL total.
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 μL assembly reaction and plate recovery on LB + Selective Antibiotic
Check out troubleshooting tips here
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-- Main.KateElston - 29 Jan 2018