re-pcr (2nd) different with pcr (1st) - (May/27/2008 )

I did PCR from plasmid rekombinant and get genes about 2.6kb (1 band electroforesis agarose 1%). If I re-amplification genes 2.6kbs, I get 5
bands (some electroforesis). My questions, why? I would like that someone to advice to me? PCR ampli condition is 94oC 2min [94oC 50oC 72oC, 1 minutes]30x
72oC 7min. thank you. I d appreciate it if you can help me

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1. your amplification time is too short. For 2.6 Kb amplification, you need to give 2.6 minutes each cycle. remember: 1 kb ->1 min

2. After initial denaturation step, I would also reduce the 94 denaturation and 50 annealing steps to 30 seconds or less, each.

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So are you doing a second PCR on your first PCR product? If so, it may be that you're adding too much DNA for a 2nd round of 30 cycles and you're starting to amplify up other things (ie: saturating the reaction).

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Thank's to guys on your advices.
I am sorry, amplification time I take 3 minutes.

Yes, I'm just adding 1 microlitres from first PCR product. And than I am doing re-PCR again with annealing temp 55oC. Result it consist by 5 bands 94oC 2min [94oC 1min, 55oC 1min, 72oC 3min] 30x 72oC 7min. My friends in a lab and promotors are confuse, what happend in PCR 1st and PCR 2nd. I hope you help me in my research PhD.

Thank you

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Why don't you try 25 cycles for your 2nd PCR and see what happens?
That's what I would do - always decrease cycle number if I get extra bands.
Good luck
Clare

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Thank you Clare, I'll try your advice
Thank a lot friends bioforum
savante

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