Two peaks in melt curve when I increase master mix volume - (Aug/18/2008 )

I was getting low Ct value and {weird curves in lower concentrations-standard curve} for this HSP gene so I increase the primer concentration by 50%. when I tested this new increase with standard curve the melt curve was fine (the master mix was for 16 wells.)

when I repeat this with 64 wells I get two peaks with every sample except the no- template control (NTC). I am sure i didn't change or make new reagent from the first test run. Only thing different was that the master mix was in the fridge for much longer than first run. I use DMSO but this I used in the test run as well.

Any ideas?? Please.

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Hi all,

I ran a agarose gel2%, and I found that ther is a secondary/non specific product formation. the non specific product is double the size of expected product and seem to increase with lower dilution of template.

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Is it from genomic contamination? What size amplicon do you expect if you amplify from gDNA?

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I really dont know. I just design the primers from mRNA sequence using primer express. I dont think there is genomic contamination. the expected product size is 63bp for cDNA

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Can you give more details, e.g. Mastermix / Taq polymerase used, Hot-Start?, pcr protocoll, primer concentrations, .......?

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