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.