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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 $\displaystyle X= (4600 / 209 ) * 0.013 * 20 = 5.72$

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

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

 Reaction 1 (CMV) 2 (CMV) 3 (HPK) 4 (HPK) Thermal Cycling Reaction Conditions 20 fmol of each DNA part (µL) 1+1 1+1 1+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) 1 1 1 1 T4 ligase (NEB) (µL) 0.25 0.25 0.25 0.25 BSMBI (µL) 0.5 0.5 0.5 0.5 dH2O (µL) 6.25 6.25 6.25 6.25 Total (µL) 10 10 10 10
• 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.