Full length cDNA - (Sep/14/2011 )
How to get full length cDNA by using PCR? in the normal way?
I try to design primers in the end of desire gene but still there are some base pares out of primer sequence,
and if i try to make the primer start from the first base but the quality of it will not be acceptable
Do you have any suggestion..
Thank you in advance
U can design a pair of primers that bind to the vector specific sequence that flank your insert site. And after PCR , u can use this as a probe to rescreen your cDNA library to increase the chance of having a full length cDNA clone
Check out a protocol known as RACE - Rapid Amplification of cDNA Ends.
thank you MurphysunHKU for your reply,
I think this kit is not cheap, also can run 10 reactions only.. Is it?
Kits aren't cheap, but buying the components and looking up the protocols is relatively cheap. Basically the kits are just a PCR kit with primers and maybe a terminal transferase. There are two sorts of RACE, 5' and 3'. 3' is much easier than the 5', and wikipedia describes the basic process and differences reasonably well.