Haynes Lab:Notebook/Short Projects/2015/02/24

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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

  1. 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.
  2. SKNSH C001, 1:1000 cDNA dilution; same.
  3. K562 C001, 1:1000 cDNA dilution; same.


Reagent Rxn1 Rxn2 Rxn3 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
Hover name
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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