User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/25

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PcTF Subcloning in E. coli Main project page
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Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Second 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) 2 (CMV) 3 (HPK) 4 (HPK) Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL)1+11+11+1 1+1
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
10x T4 ligase buffer (Promega) (µL)111 1
T4 ligase (NEB) (µL)0.250.250.250.25
BSMBI (µL)0.50.50.50.5
dH2O (µL)6.256.256.256.25
Total (µL)10101010
  • Bacterial transformation , Long transformation protocol
    • Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.


No colony on plates with BL21 strain. and 100s of colonies on BD003 and BD004 plasmids transformed in DH5α.

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