User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17

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 Desired Constructs: BD003: 5XGAL4-Spacer-CMV-Kozak-AMCyan-AmCyan-NLS-Stop BD004: 5XGAL4-Spacer-HPK-Kozak-AMCyan-AmCyan-NLS-Stop Existing construct is: 5XGAL4-Spacer-HsvTK-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 mammalian vector Amplification of CMV and HPK from KAH187 and KAH184 respectively. The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though Site Directed Mutagenesis PCR Reactions Part BD002 HPk CMV Reaction Conditions Gel Picture DNA Template (µL) 0.1 0.1 0.1 95°C, 3 min. [95°C, 30 sec.; 45°C, 40 sec.; 72°C, 6 min.] x35 72°C, 3 min. 4°C, ∞ Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps Forward Promer (µL) 1 1 1 Reverse Primer (µL) 1 1 1 2X GoTag (µL) 50 50 50 dH2O (µL) 47.9 47.9 47.9 Tolal (µL) 100 100 100 Extracting linearized plasmids with Sigma PCR DNA Purification Kit DNA Part 260/280 ng/µL CMV 1.9 42 HPK 1.8 70 Backbone Plasmid 1.8 209 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= (2203 / 70 ) * 0.013 * 20 = 8.18 }[/math] CMV [math]\displaystyle{ X= (2275 / 42 ) * 0.013 * 20 = 14.08 }[/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. Result 100s of colonies on plates with DH5α , No colonies on plates with BL21.