Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29

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1/29/13

Type IIS Assembly Day 2


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

Purified PCR Product to 20fmol/μL

Sample μL of Sample μL of dH2O
1. pSB1A3 7.8 12.2
2. hPCD 2.8 17.2
3. BL02 6.4 13.6
4. BL03 5.5 14.5
5. BL04 8.8 11.2
6. fshPCD 2.3 7.7
  • 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 (1 μL) 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
Reagent 1 2 3
gg2 pSB1A3 1.0 1.0 1.0
gg3 hPCD 1.0 1.0 1.0
BL02 1.0 --- ---
BL03 --- 1.0 ---
BL04 --- --- 1.0
10x ligase buffer 1.0 1.0 1.0
NEB T4 lgase 0.25 0.25 1.0
BsmBI 0.5 0.5 0.5
dH2O 5.25 5.25 5.25
Total 10.0 10.0




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