Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Third 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)BD003
|
2 (HPK)BD004
|
3 (-)Ctrl Vector Only
|
4 (-)Ctrl CMV Only
|
4 (-)Ctrl HPK 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.
- Decrease the temperature stepwise from 80°C to 4°C
- 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
|
Result (Colonies) |
8 |
NO |
~50 |
~80 |
~100
|
- Bacterial transformation , Fast transformation protocol
- Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 20 min.. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
No colony on plates for BD003. and 8 of colonies on BD004, and BD004 plasmids transformed in DH5α.
|