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cloning short genomic DNA fragments

gDNA cloning intron pcr

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#1 lb8x



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Posted 16 August 2012 - 06:44 AM

We need to clone relatively small (1.5 kbp) fragments of human genes. They contain both intron and exon regions. Because they are not big, we have tried to simply clone them in good-old pBluScript and into another vector as well. The fragments are amplified by PCR with restriction sites inserted near the extremities - nothing groundbreaking, it's the same approach that we are using all the time for cloning cDNAs. We are trying to clone 2 different gDNA fragments and nothing worked.
So we are wondering:
1. Does it take a special type of plasmid to clone gDNA fragments? (perhaps due to some sequences in the introns)
2. Does it take a special strain of E coli to grow such plasmids?
Thanks for any helpful advice you would have

#2 bob1


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Posted 16 August 2012 - 12:56 PM

The answers are no, and no. It should work fine, just make sure that your ligase is fresh (fresh buffer too - the ATP goes off), and that you are sure that your digestions are working.

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