04/01/15 - Alexander Ellingson
PCR-verify activation domain primers
Try different cDNA libraries for 12 primer fragments to account for any mutations or deletions. First 12 fragments run to verify for any variations. iPS cell template utilized in place of U20S, SKNSH, and K562 templates.
- iPS, 1:100 cDNA dilution; CARM1 = 1827 bp: CARM1 F1/ CARM1 R1; MYB = 159 bp: MYB F1/ MYB R1, KMT2A = 1134 bp: KMT2A F1/ KMT2A R1; KMT2C = 423 bp: KMT2C F1/ KMT2C R1; KMT2D = 423 bp: KMT2D F1/ KMT2D R1, KMT2E = 1176 bp: KMT2E F1/ KMT2E R1; CBP = 2031 bp: CBP F1/CBP R1; KAT2B = 1953 bp: KAT2B F1/KAT2B R1; SETD1A = 420 bp: SETD1A F1/SETD1A R1; SETD1B = 420 bp: SETD1B F1/SETD1B R1; SETD7 = 459 bp: SETD7 F1/SETD7 R1.
| Reagent
|
Rxn1
|
Rxn2
|
Rxn3
|
Expected:
1. CARM1 = 1827 bp
2. P300 = 1848 bp
3. MYB = 159 bp
4. KMT2A = 1134 bp
5. KMT2C = 423 bp
6. KMT2D = 423 bp
7. KMT2E = 1176 bp
8. CBP = 2031 bp
9. KAT2B = 1953 bp
10. SETD1A = 420 bp
11. SETD1B = 420 bp
12. SETD7 = 459 bp
13. SMCA4 = 4428 bp
14. ANM5 = 1911 bp: ANM5 F1/ ANM5 R1;.
|
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 ∞
|
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