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High Molecular weight artifact LA-PCR 2-step - Artifact or real band - higher than expected Mw amplified product (Oct/22/2009 )

I am carrying out a mouse mitochondrial DNA amplification using the one-shot LA-PCR reaction by Takara. I started with 100ng template, than 50ng and even at 1ng, with primers for a 14.3kB product. Here's the problem I face, I do a 2-step PCR with 2min 94C initial Denature, then 26 cycles of 98C 10sec, annealing and extension 10min at 68C, final extension 72C 10min (this is as per the 2-step protocol by Takara. I end up with a very high molecular weight amplified product ~ 30kB. What could be going wrong. The primers are right and show 100% homology with blastn. The Tm for the primers are 64 and 68C. I even introduced a annealing step at 63C for 20sec in the cycle but same result. If someone could help me resolve this issue I really appreciate that. Thank you in advance.
Sharuc

-Sharuc-

hi tehre.. can you also tell what were your controls? sounds strange at the first look though!!!
i also feel the extension step is too long for even a 14.3 kb product... have you done it on a Q-PCR?

-Pradeep Iyer-

The controls are good, theres no template control, no primer control, I don't see any bands in these. The extension time is based on takara suggestion, they use 15min for a 17.3kb product at 68C. No did not do a qPCR.
Thanks for helping out.

-Sharuc-

Sharuc on Fri Oct 23 11:40:47 2009 said:


The controls are good, theres no template control, no primer control, I don't see any bands in these. The extension time is based on takara suggestion, they use 15min for a 17.3kb product at 68°C. No did not do a qPCR.
Thanks for helping out.


Have you had any luck with the TaKaRa LA-PCR amplification yet? I am also having issues getting good amplification of a 13 kb segment using TaKaRa.

-Zach T-