long size cDNA cloning - (Jan/10/2007 )
Hi to everyone,
I need advise concerning cDNA cloning. I want to clone into an expression vector the cDNA from a gene of 15 kb (yes, really small one). I guess it is quite difficult, first to get full length cDNA of this size and afterwards to amplify and clone.
At the moment there are no commercial clones (Origene, RZPD).
How can I proceed? Anyone tried before such a big sized cloning?
And I don´t have enough budget to synthesis the clone
I suspect your idea of amplify such big sequence, it might be amplified, but think about the chances of mutant introduced and also it is very difficult to find it by sequencing in such big fragment.
if possible, you mght design experiment uisng RE digestion subcloning
I have clone >20kb fragment to SK from lambda DNA clone (screened from genomic DNA library), the fragment was digested by NotI (no Not found in this fragment)and SK was also digested by NotI and dephorosphorated.