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

CTLA4

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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 product page 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)