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PCR of repeated region


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#1 tatzilo

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Posted 14 January 2011 - 08:28 AM

Hi there,

I'm trying to amplify a gene region which is duplicated. Instead of the whole region (1 kb) I only get the first part (0.5 kb).
Unfortunately both regions end with almost the same bases (4 changed out of 20).

Any ideas which variables I could change? No success with an alteration in template concentration. Next I'd like to play with temperature,
maybe run a gradient. What else could I try?

Thanks for brainstorming!

#2 phage434

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Posted 14 January 2011 - 10:41 AM

You will probably have to redesign your primer. Two primers which differ by a single base at the 3' end will in general not hybridize to the same location. Try to make a primer for your longer fragment which mis-matches the shorter fragment by at least the 3' base.




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