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PCR produces products of wrong size


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4 replies to this topic

#1 gorkin

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Posted 26 March 2009 - 07:43 AM

Hi, im trying to clone two genes coding for a similar protein from a parasite. Theres supposed to be no introns in my gene. My problem is that the primers are exactly complementary to other regions of the genome (which is extremely weird in my opinion). Thus its quite hard to clone the genes I want, I just have tried and tried and it didnt produce. I finally got some inserts to ligate today and did a digestion analysis...my gene is supposed to be around 2kb and I got 1 kb long products. What should I do to clone these genes? I cant just make different primers, as I need the gene from start to finish with added restriction sites and proteolysis site at the end.

#2 gorkin

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Posted 26 March 2009 - 07:50 AM

Uh, I forgot to add that I got around the correct size product direct from PCR, but the genes my primers are complementary with produce similar size products. And so after digestion with NcoI- or NdeI-XhoI, I had the problem I mentioned in the above post.

#3 bochum

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Posted 27 March 2009 - 04:59 AM

I bit confused with your question but if you have such problems like same completementrary seq and product size of 2kb.it looks difficult so better do divided this 2kb into 1kb and subclone then clone them back.
With Regards
Martin
Career In Life Science

#4 mastermi

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Posted 28 March 2009 - 05:31 AM

It might be better to amplify a bigger fragment first (with specific primers), ligate this into a vector and do a PCR with the primers you need on the ligation product.

#5 gorkin

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Posted 29 March 2009 - 02:55 AM

Thanks for the suggestions!




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