User:Tim Henry/Notebook/CTLA4/2012/01/28

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CTLA4 exon 1 +49 A/G SNP

  • Retrieved primer via various journal articles
    • 5'-GCTCTACTTCCTGAAGACCT-3'
    • 5'-AGTCTCACTCACCTTTGCAG-3'
  • Purchased BbvI restriction enzyme
  • Use templates of family (4)
  • Attempt to genotype A/G for SNP
  • Result on first attempt was ruined by primer dimer

Reaction

  • 2 μL template
  • 1 μL primer (x2)
  • 16 μL dH2O
Initial 94°C 4m
Denature 94°C 20s
Anneal 57°C 15s
Extend 72°C 30s
Final 72°C 10m

30 cycles

Digestion

2 μL BbvI 2 μL NEB 2 Buffer

1μL is listed as the "normal" amount. I thought 2 would go faster. Buffer concentration was a complete guess. It says the glycerol percentage should not be greater than 5% of total volume. 20μL before (pcr rxn), and 24μL with enzyme and buffer. Cannot tell if digestion would have worked or not. Ideally I'd run the pcr, electrophoresis. Run pcr+digest, electrophoresis.

Intended to digest 1 hr, didn't realize time and stopped at about 75min though.

Looking at journal articles again I remember see 2hr digestion mentioned at least once. But data sheet implies digestion over 1hr is not advised.

Resulting Gel

Likely primer dimer based on 40bp result. Expected band locations for associated alleles overlaid.

HyperLadder V (25-500bp)

Thenry 01282012 CTLA4 49.jpg