User:Daniel A. Vargas/Notebook/General lab notebook/2015/07/21

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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
Damaged Gel
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
PCR 7/21
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.