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I am using PCR to amplify a 1000bp region of the CytB mt gene. Some of the samples had not amplified and I wish to change the reaction components (or use additives like BSA) to attempt at getting amplification. The DNA was extracted from liver/muscle samples, so I am not expecting many inhibitors present, but I am eluting samples in AE buffer.

I used the following combinations of PCR mixture but the reactions still failed (reaction volume 20ul)

Combination1 : MasterMix (10ul), Primer 1 (1ul), Primer 2 (1ul), BSA 2mg/ml (2ul), water (4ul), DNA (2ul) Combination2 : MasterMix (10ul), Primer 1 (1ul), Primer 2 (1ul), BSA 2mg/ml (4ul), water (3ul), DNA (1ul) : Tried to increase BSA concentration and reduce DNA to decrease the volume of inhibitors (if any) going into the reaction mixture.

Someone who has previously used BSA to successfully troubleshoot PCRs, am I going about this in the right way ? If I need to take a different approach please help me out.

Thanks!

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  • $\begingroup$ The albumin shouldn't be what your trouble shooting I don't think. It's something I've always kept constant if required by the enzyme. Since your inconsistency is across different samples and I assume you are creating a 'mastermix' containing the taq, ntp's, primer etc I would troubleshoot the purification steps. If that's not possible, there are other parameters of The pcr (especially annealing temp and primer concentration) that will have more apparent results. MtDNA usually requires a more stringent extraction protocol like using guanadine. $\endgroup$ – rhill45 Aug 19 '15 at 13:10
  • $\begingroup$ There are other samples from the same extraction batch that are working fine, so I'm assuming the problem is not with the extraction or with the PCR temperature settings ? And I use a mastermix, yes. $\endgroup$ – Anurag Mishra Aug 19 '15 at 16:28

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