1 reply to this topic

[Rubicon]

Hi guys!
I am a bit confused since my new project requires to perform a cloning of a huge human gene consisting of 31 exons, all together they are about 8 kb BUT!!! The problem is that exons are separated by introns which are more than 10kb each. Therefore, the only possibility I see right now is to subclone all of 31 exons individually and afterwards ligate them which might be extremely time consuming procedure... Would it be an another possibility to get full length cDNA of this gene? currently I have only the genomic DNA from this sample...
Thanks in advance for your help!

Edited by Rubicon, 15 February 2011 - 08:02 PM.

[phage434]

PCR on the cDNA would be the first strategy to try.  Given it is 8Kb, you might sensibly do the PCR on smaller, overlapping regions, then splice those back together, since lengthy pcr reactions from cDNA are problematic.  Or you could simply get it synthesized.