My expected amplicon size is 7.6 Kb. I am getting a very faint band of this size along with other non specific bands. I have tried every possible condition to increase the intensity of my band but could not. As the band intensity is low I cant proceed with further downstream steps. I tried to gel extract my desired band but the concentration is 20 ng/ul. Can I use this extracted pcr product as template for the second round of PCR under same conditions and what should be the dilution factor??
I would read the manual for your PCR enzyme (the suggested DNA amount) and at least once try the reaction.
Here are the things to consider:
1. Assuming you're using a reliable proof-reading polymerase in both PCRs, there's still a chance of getting doubled sequence deviations.
2. Some polymerases need extra DNA space prior to the primer to start the reaction.
3. PCR conditions put a strain on the DNA, which might result in crumbled ends. Assuming you're going to cut out your target sequence out of the 7,6kb, this should not be a problem.
I tried yesterday, did not work for me. got a smear....
Platinum TAQ DNA polymerase kit, Catalogue Number: 10966-026 (250 x 25 µl reactions)
Hey Zohaib,
What was the concentration of template you used in your reaction?
Yes. Use Q5 High-Fidelity DNA Polymerase (NEB) or kod high fidelity DNA polymerase (toyobo) for second round of PCR.
Hi,
I think you can try to amplify the PCR product extracted from the gel, but I suggest you to optimize your PCR first. Is the starting template ok? I mean, pure and not degraded. Please digest it with the proper restriction enzyme and run the digested product on a gel to ensure that you're starting form a good template. Then try to check and adjust the PCR conditions.
Have a nice day
Hi Farnaz,
You can use your purified PCR product as a template for 2nd round of PCR reaction. Please use either 1ul/2ul/3ul of ( in three separate reaction) purified PCR product for 2nd round of reaction. You can use Phusion polymerase, however, if possible please use KOD- Hot Start DNA polymerase. All the best!
I agree with the relevant suggestions given above. If you are still not able to fix your problem, you can use low amount of template DNA and amplify for 35 cycles. You will get good amounts. In addition, you can use PCR purification kits instead of Gel extraction kits for increasing the yield of purified PCR products for performing further downstream procedures.
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