4 replies to this topic

[noyara]

Hi
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
Noor

[MurphysunHKU]

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

[bob1]

Check out a protocol known as RACE - Rapid Amplification of cDNA Ends.

[noyara]

thank you MurphysunHKU for your reply,


Hi bob1,
I think this kit is not cheap, also can run 10 reactions only.. Is it?

[bob1]

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.