Your Name - dd/mm/yy
PCR-verify activation domain primers
Try different cDNA libraries for the CARM1 and MYB fragment to account for any mutations or deletions
- U2OS C002, 1:1000 cDNA dilution; KMT2A = 1134 bp: KMT2A F1/ KMT2A R1; KMT2C = 423 bp: KMT2C F1/ KMT2C R1; KMT2D = 423 bp: KMT2D F1/ KMT2D R1.
- SKNSH C001, 1:1000 cDNA dilution; same.
- K562 C001, 1:1000 cDNA dilution; same.
| Reagent
|
Rxn1
|
Rxn2
|
Rxn3
|
Expected:
1. U2OS KMT2A = 1134 bp
2. SKNSH KMT2A = 1134 bp
3. K562 KMT2A = 1134 bp
4. U2OS KMT2C = 423 bp
5. SKNSH KMT2C = 423 bp
6. K562 KMT2C = 423 bp
7. U2OS KMT2D = 423 bp
8. SKNSH KMT2D = 423 bp
9. K562 KMT2D = 423 bp
|
10 μL/lane, 1% agarose; Ladder
|
| Template |
1.0 |
1.0 |
1.0
|
| 10 uM fwd primer |
1.0 |
1.0 |
1.0
|
| 10 uM rev primer |
1.0 |
1.0 |
1.0
|
| 2x GoTaq green |
12.5 |
12.5 |
12.5
|
| dH2O |
9.5 |
9.5 |
9.5
|
| |
25.0 |
25.0 |
25.0
|
Program: GOTAQ35cyc
- 95°C, 3 min
- 35x[95°C, 30 sec; 57°C, 30 sec; 72°C, 2 min]
- 72°C, 5 min
- 4°C ∞
- primers 4-6 attempted in order to rule out the possibility of primer discrepancy; the camera is damaged, so manual imaging techniques are necessary.
|
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