10/10/12
Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene
- Template strands are about 7kb for BD002.
- Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μM concentration.
- Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration.
- DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut.
- Serial dilution of template to: 1. 100 ng/μL 2. 80 ng/μL 3. 10 ng/μL 4. 1 ng/μL 5. 200 pg/μL
Reagents |
1 |
2 |
3 |
4 |
5
|
Plasmid DNA |
0.2 μL |
0.5 μL |
0.5 μL |
0.5 μL |
1 μL
|
primer 1 (10 μM, 125 ng) |
1 μL |
1 μL |
1 μL |
1 μL |
1 μL
|
primer 2 (10 μM, 125 ng) |
1 μL |
1 μL |
1 μL |
1 μL |
1 μL
|
2X GOTAG (PCR Master Mix) |
25μL |
25μL |
25μL |
25μL |
25μL
|
dH2O |
22.8 μL |
22.5 μL |
22.5 μL |
22.5 μL |
22 μL
|
Total |
50 μL |
50 μL |
50 μL |
50 μL |
50 μL
|
- No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
- Thermal Cycling
- 95°C/ 30 sec
- [95°C/ 30 sec; 55°C/ 1 min; 72°C/ 6 min (1 min/kb plasmid length)]x30 cycle
- 4°C, ∞
- Run 5 μL of each PCR reaction on 1% agarose gel.
- DNA Purification Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products.
- The purified result: Column1: 100ng/μL and Column 3: 28ng/μL.
- Cutting the SAPI sites to make the sticky ends for ligation:
Amplified DNA (500ng) |
5.00 μl |
17.00 μL
|
SAPI |
1.0 μl |
1.0 μL
|
10x buffer |
5.00 μl |
5.00 μL
|
dH2O |
39.00 μl |
27.00 μl
|
Total |
50.0 μl |
50.0 μl
|
Incubate at 37°C overnight.
|
- Restriction enzyme deactivation Incubate the tubes at 80°C for 10 minutes and cool down gradually in room temperature.
- Ligate The Sticky Ends to Make Circular DNA
DNA Conc. |
10ng |
20ng |
30ng |
50ng |
50ng (Ctrl)
|
DNA μL |
1μL |
2μL |
3μL |
5μL |
5μL
|
T4 Ligase |
1.0 μl |
1.0μL |
1.0μL |
1.0μL |
0.0μL
|
2X Roche Buffer |
5.00 μl |
5.00 μL |
5.00 μL |
5.00 μL |
5.00 μL
|
dH2O |
5.00 μl |
5.00 μl |
5.00 μL |
6.00 μL |
0.00 μL
|
Total |
10.0 μl |
10.0 μl |
10.0 μl |
10.0 μl |
10.0 μl
|
Incubate at room temperature for 30 minutes.
|
Transformation result |
No colony |
No colony |
No colony |
Two Colonies |
No Colony
|
- Long transformation protocol in BL-21.
- Grow on AMP agar plates and incubate overnight at 37°C.
Mutagenesis Confirmation
- Grow the colonies in LB broth for 6 hrs.
- Miniprep the plasmids, final concentration: 491ng/μL
Plasmid DNA (BD002) |
1 (4.0 μl) |
2 (4.0 μl) |
3 (4.0 μl) |
4 (4.0 μl) |
5 (4.0 μl)
|
BSMBI |
1.0 μl |
--- |
--- |
--- |
1.0 μl
|
NotI |
--- |
1.0 μl |
1.0 μl |
--- |
1.0 μl
|
SpeI |
--- |
1.0 μl |
--- |
--- |
---
|
10x FastDigest buffer + green loading dye |
1.5 μl |
1.5 μl |
1.5 μl |
1.5 μl |
1.5 μl
|
dH2O |
9.5 μl |
7.5μL |
9.5μL |
10.5μL |
7.5μL
|
Total |
15.0 μl |
15.0 μl |
15.0 μl |
15.0 μl |
15.0 μl
|
Incubate at 37°C for 30 minutes.
|
- Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.
Sequencing Analysis
Mutated cut site is marked with red.
|