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 PcTF Subcloning in E. coli
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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 [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
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1 (CMV)
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2 (CMV)
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3 (HPK)
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4 (HPK)
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Thermal Cycling Reaction Conditions
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| 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, ∞
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| 10x T4 ligase buffer (Promega) (µL) |
1 |
1 |
1 |
1
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| T4 ligase (NEB) (µL) |
0.25 |
0.25 |
0.25 |
0.25
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| BSMBI (µL) |
0.5 |
0.5 |
0.5 |
0.5
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| dH2O (µL) |
6.25 |
6.25 |
6.25 |
6.25
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| Total (µL) |
10 |
10 |
10 |
10
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- 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.
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PcTF Subcloning in E. coli
