02/24/15 - Alexander Ellingson
PCR-verify activation domain primers
Try different cDNA libraries for the CARM1, P300, and MYB fragment to account for any mutations or deletions
- U2OS C002, 1:1000 cDNA dilution; CARM1 = 1827 bp: CARM1 F1/ CARM1 R1; P300 = 1848 bp: P300 F1/ P300 R1; MYB = 159 bp: MYB F1/ MYB R1.
- SKNSH C001, 1:1000 cDNA dilution; same.
- K562 C001, 1:1000 cDNA dilution; same.
Reagent
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Rxn1
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Rxn2
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Rxn3
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Expected: 1. U2OS CARM1 = 1827 bp 4. U2OS P300 = 1848 bp 7. U2OS MYB = 159 bp 2. SKNSH CARM1 = 1827 bp 5. SKNSH P300 = 1848 bp 8. SKNSH MYB = 159 bp 3. K562 CARM1 = 1827 bp 6. K562 P300 = 1848 bp 9. K562 MYB = 159 bp
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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, 1 min; 60°C, 1 min; 72°C, 3 min]
- 72°C, 3 min
- 4°C ∞
- Note: the cycler was to run at 57°C for the annealing segment, but has instead run at 60 as someone reprogrammed it.
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