# Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29"

Engineering PC-TFs <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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.
 Reagent Vol. Thermal cycling [45°C, 2 min.; 16°C 5 min.] x25 60°C, 10 min. 80°C, 20 min. 4°C, ∞ 20 fmol of each DNA part up to 8.0 10x T4 ligase buffer (Promega) 1.0 T4 ligase (NEB) 0.25 BsmBI 0.5 dH2O 0.25 10.0 μL

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