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 Engineering PC-TFs
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Summary
Digestion/ ligation reaction
Dilute the purified PCR product to 20 fmol/μL- Measure ng/μL of the purified sample.
- The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
- Formula: x = length in bp ÷ measured ng/μL * 0.26
- Perform BsmBI/ T4 ligase mediated assembly
- BsmBI cuts the DNA fragments and creates complementary overhangs.
- Complementary sticky ends anneal via base pairing.
- T4 ligase seals gaps in the phosphodiester DNA backbone.
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Reagent
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Vol.
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Thermal cycling
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
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| 20 fmol of each DNA part |
up to 8.0
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| 10x T4 ligase buffer (Promega) |
1.0
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| T4 ligase (NEB) |
0.25
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| BsmBI |
0.5
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| dH2O |
0.25
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10.0 μL
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Engineering PC-TFs
