Plasmid Verification
| Reagent
|
Volume
|
Expected:
1. ANM5 = 4269,
2. MYB = 4,483
3. Carm1 = 4,418
4. SETD7 = 5,112
5. KMT2E = 4,418
6. SETD1A = 7,715
|
10 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) 1:100 |
3.0 μL
|
| 6X BioLabs purple dye |
3.0 μL
|
| dH2O |
4.0 μL
|
| |
10 μL
|
Mini prep Results
| Plasmid
|
Conc. (ng/µL)
|
St Dev (ng/µL)
|
260/280
|
| ANM5 |
312.5 |
4.13 |
1.89
|
| MYB |
286.528 |
3.20 |
1.90
|
| CARM1 |
381.112 |
5.13 |
1.87
|
| SETD7 2** |
24.418 |
3.63 |
1.98
|
| KMT2E |
277.615 |
4.23 |
1.90
|
| SETD1A |
222.234 |
2.56 |
1.92
|
Gel electrophoresis of PCR amp
| Reagent
|
Volume
|
Expected:
1. ANM5 = 1911,
2. MYB = 159
3. Carm1 = 1827
4. SETD7 = 459
5. KMT2E = 1176
6. SETD1A = 420
|
15 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) 1:100 |
1.0 μL
|
| 1X GoTaq |
12.5
|
| F primer |
1.0
|
| R Primer |
1.0
|
| dH2O |
9.5
|
| |
25 μL
|
Program: Gotaq35cyclo (block A lane4,5)
- 95°C, 3 min
- 35x[95°C, 30 sec; 52°C, 30 sec; 72°C, 120 sec]
- 72°C, 5 min
- 4°C ∞
Program: Gotaq35cyclo (block B 1-3, and 6)
- 95°C, 3 min
- 35x[95°C, 30 sec; 58°C, 30 sec; 72°C, 120 sec]
- 72°C, 5 min
- 4°C ∞
Conclusion
There is no plasmid in the lab for the SETD7 so Embryonic stem cell cDNA was used to try to obtain a product. For all other active domains the product from the mini prep was used for pcr. Lane 3, which is CARM1, will need to be ran at a higher temperature due to the excess amount of nonspecific binding products. The CARM1 PCR will be ran at 64, 68, and 70 degrees Celsius, closer to the Tm of the primers. All other reactions gave bands that were expected and will be ready for the Phusion protocol.
|
Project name
Main project page
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