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

PcTF Subcloning in E. coli

Main project page Previous entry      Next entry

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 [math]\displaystyle{ X= (4600 / 209 ) * 0.013 * 20 = 5.72 }[/math] HPK [math]\displaystyle{ X= (516 / 70 ) * 0.013 * 20 = 1.91 }[/math], [math]\displaystyle{ 1.91 + 18.1 = 20 }[/math] CMV [math]\displaystyle{ X= (588 / 42 ) * 0.013 * 20 = 3.64 }[/math], [math]\displaystyle{ 3.64 + 16.36 = 20 }[/math] 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. No colony on plates with BL21 strain. and 100s of colonies on BD003 and BD004 plasmids transformed in DH5α.