3 replies to this topic

[Biog]

Hi all,

I would like to know which is more efficient and cheaper to use in molecular cloning: TOPO (Invitrogen) or pGEM-T (Promega) ?
Or is there another new technique, which is better than the two previous ones?

Thanks,

[Rsm]

I found pGEM cheaper and as efficient as TOPO.
However, it depends what you want to do with your gene. If you want to overexpress it, you can directly clone it into your expression plasmid. That saves you much more time and money.

rsm

[Biog]

Rsm, on 02 December 2010 - 12:58 AM, said:

I found pGEM cheaper and as efficient as TOPO.
However, it depends what you want to do with your gene. If you want to overexpress it, you can directly clone it into your expression plasmid. That saves you much more time and money.

rsm

Thank you for your answer.
You mean cloning directly into expression plasmid without intermediary cloning in pGEM-T?

Edited by Biog, 02 December 2010 - 08:47 PM.

[Rsm]

Yes, make PCR primer
FORWARD: containing some 5' T, then a specific restriction enzyme site, Kozak sequence, and the first 20nt of your gene (like for example for GFP tttGAATTCgccaccATGGTGAGCAAGGGCGAG)
REVERSE: again, some 5'T, RE site, stop codon and 20nt of your gene, like TTTggatccTTACTTGTACAGCTCGTCCATGC (don't forget to reverse complement!)
and do a PCR using a proof-reading enzyme. Then PCR cleanup (columns), digest, gel cut, ligate and transform.

Works in 90% of the cases...

rsm