User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/29

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Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Third Try)

  • 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 = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
    • Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20

Backbone vector X = (4600 / 209) * 0.013 * 20 = 5.72

HPK X = (516 / 70) * 0.013 * 20 = 1.91, 1.91 + 18.1 = 20

CMV X = (588 / 42) * 0.013 * 20 = 3.64, 3.64 + 16.36 = 20

Reaction 1 (CMV)BD003 2 (HPK)BD004 3 (-)Ctrl Vector Only 4 (-)Ctrl CMV Only 4 (-)Ctrl HPK Only Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL)1+11+11 1 1
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • Decrease the temperature stepwise from 80°C to 4°C
  • 4°C, ∞
10x T4 ligase buffer (Promega) (µL)111 1 1
T4 ligase (NEB) (µL)1.01.01.01.0 1.0
BSMBI (µL)0.50.50.50.5 0.5
dH2O (µL)5.55.56.56.5 6.5
Total (µL)10101010 10
Result (Colonies) 8 NO ~50 ~80 ~100


  • Bacterial transformation , Fast transformation protocol
    • Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 20 min.. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.


No colony on plates for BD003. and 8 of colonies on BD004, and BD004 plasmids transformed in DH5α.

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