This PCR is inverse PCR product. The DNA was digested and ligated and PCR was conducted on circular piece of DNA.
It is often necessary to ligate many different amounts of dna in the ligation step to find the best concentration for circularisation rather than linear ligation of restriction fragments. possibly also the circularised dna is too large for the pcr conditions to work well. possibly use a longer extension time
It is often necessary to ligate many different amounts of dna in the ligation step to find the best concentration for circularisation rather than linear ligation of restriction fragments. possibly also the circularised dna is too large for the pcr conditions to work well. possibly use a longer extension time
according to your gel picture it has been depicted that primer dimer has been formed only, u should quantify your template before putting any reaction.. min 100ng of template/20ul of PCR reaction should be kept...
Thank you for all the answers, I diluted my DNA further to see if it works and also reduced my number of cycles. I am waiting for my gel results.
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