User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/06

PcTF Subcloning in E. coli

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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: ~5500 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 Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. PCR product. Incubate at 37°C for 15 min. Note: DpnI cuts the methylated template DNA, not the unmethylated Reagent Vol. DpnI Digest 37°C, 15 min. PCR reaction 95.0 μL 10x FD buffer (Fermentas) 5.0 DpnI (Fermentas) 1.0 dH2O ---   102.0 μL Extracting linearized plasmids with Sigma PCR DNA Purification Kit DNA Part 260/280 ng/µL CMV 1.9 55.0 HPK 1.9 115.0 Backbone Plasmid 1.86 170 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= (5500 / 170 ) * 0.013 * 20 = 8.4 + 11.58 = 20 }[/math]μL HPK [math]\displaystyle{ X= (2203 / 115 ) * 0.013 * 20 = 1.16 + 19 = 20 }[/math]μL CMV [math]\displaystyle{ X= (2275 / 55 ) * 0.013 * 20 = 2.78 + 17.2 = 20 }[/math]μL Reaction 1 BD003(CMV) 2 BD004(HPK) 3 (-) Ctrl(HPK Only) 4 (-) Ctrl(CMV Only) 4 (-) Ctrl(BD002 Only) Thermal Cycling Reaction Conditions 20 fmol of each DNA part (µL) 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 1 T4 ligase (NEB) (µL) 1.0 1.0 1.0 1.0 1.0 BSMBI (µL) 0.5 0.5 0.5 0.5 0.5 dH2O (µL) 5.5 5.5 6.5 6.5 6.5 Total (µL) 10 10 10 10 10 Bacterial transformation , Long transformation protocol Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 30 min., Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.