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.
Hello Nalini,
I expect you have the answer by now but for others reading this thread:
your result is as expected. You must realise that pcr doubles the amount of product every cycle so every 10 cycles you have 1000 times more product than you started with. You have used too much product for the second round of amplification and the product is soon at very high concentration and self annealing to other product molecules so the product is getting randomly longer and producing the smear of high molecular weight material that you see on the gel.
You should dilute 1ul of product in 100ul of water and run about 6 tubes of pcr with 1ul of this dilution as template. remove 1 tube from the pcr machine every 5 cycles and probably one of the tubes at 10-15 cycles will have lots of quite clean amplimer and the 20-30 cycle tubes will look like your result with just a smear. The best way to get clean product in this example is to us e2 nested primers just inside of your first round primers.
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