Dear All,
I'm now trying to amplify a gene with 3690bp from the genomic DNA. I've design 3 sets of primers. First set is flanking the whole gene. Second set is flanking from 1 to 2613bp. Final set is flanking from 2614 to 3690bp. I can only amplify sequence from 2614 to 3690bp but fail to amplify the other 2.
PCR cycles is 98oC 3 mins 98oC 40s 55oC 30s 75oC 2min 72oC7min (35 cycles) using Pfu polymerase.
Genomic DNA was prepared by lysing bacteria cell pellet with 20ul 0.5M NaOH and 0.25% SDS, then boil in 100oC for 10min and diluted lysate with 200ul water.
PS. the GC content of the gene is around 38%. Will adding DMSO be helpful?
Could anyone provide some suggestion for my amplification?
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