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, ∞
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
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.