Hey everyone, this might sound like a call for help due to laziness, but we tried now several different primer pairs covering the last coding exon of CACNG8 (in mouse and rat). The overall goal is to sequence the amplicons after CRISPR, but this gene is very special as it seems. We assume that the very high GC content in exonic and intronic sequences hampered our trials. Maybe someone working on this particular gene has some functional primers (or tipps concerning this locus) he or she could share with us?
Thank you very much in advance and have a wonderful time in the lab
If the problem is just that the template is high GC then use your hottest annealing primers ( or make them longer to anneal at 65-70c and run the pcr in final concentration of 1M betaine or up to 7% DMSO which may open up the secondary structure enough to amplify. If you can anneal the primers as hot as possible then the template can not reanneal well which increases the chance of amplification. A temperature gradient of annealing temperature in presence of betaine would be a good start. There are other additives for high GC templates but dmso and betaine are a good start
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