Dear All,
I'd like to characterize 2 mouse cell lines in terms of mutations and would like to know if you had good primers and protocol (or any other way to test it) to test the following genes:
Kras (codon 12-13-61), Braf (V600E), Pten, Egfr, Pi3kca (exon 20).
Thank you in advance for your responses.
Christophe
hi Christophe, as a bare minimum I'd suggest sequencing all five genes. If possible, you could perform WGS and/or WES on all mouse cell lines as you are likely to need it for your publication. Best, Tamara
Hi Tamara,
Thank you for your answer. Yes it is a good idea. I wanted to go for a cheaper option but might be in the end the best solution!
Best
Christophe
Hi Chistophe, you would have to 1. isolate DNA from each cell line, 2. PCR to amplify the region of interest, and 3. send sample for sequencing. You will have to make different sets of primers for each gene (total 10 primers, accounting for forward and reverse primers). If you need help with making specific primers, you can PM me, but there are lots of materials out there (Tm~60, ~20 bp, ~50% GC, 5'-heavy GC--test the primer on primer3plus prior to purchasing). Some tips in your case, make sure to design primers on the same exon and use a high fidelity polymerase, like phusion. The total cost varies, but a very conservative estimate would be ~$5 primer and ~$5 per sequence, so
2 cell lines *(5 to 10) sequencing primers + creating 10 primers = 100-150. Plus, you would have enough primer to last you a looong time in case you want to sequence other cell lines in the future.
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