I need to amplifying 3'UTR of a gene from cDNA for the reporter assay. In the first round of PCR, I have got the specific amplification at 584 bp. However when i have re-amplified the PCR product , I am not able to succeed with it. I have used 2ul of the amplified product as the template for 20ul PCR reaction. Kindly suggest me to rule out the error and the best way to amplify the 3'UTR successfully. Below is gel picture of second round PCR. Lanes followed by ladder are negative control and two different samples
When you made the first PCR was amplified small amounts of nonspecific fragments. On the second PCR, these fragments will be amplified, since its ends will be the same primers that you are using
Hi Nalini in this case it's suitable to try a gradient concentration of your PCR product in order to start your second amplification because you don't know the real concentration of the amplifia.
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