PCR Cloning - (Aug/22/2006 )

Hi....

Someone out there wud kindly help me out....
I have a 801bp PCR product amplified...want to clone it and c in E.coli.....
After reading the T/A based pcr subcloning from the protocol online site....having less idea how to go forward with it....someone who has tried this out could share with me the protocol ...that is what wud be the concentration of the amplified product to be used?? the amount of T vector??and all....iam juz a starter with cloning.....

thank you in advance

-chimmi-

Use 15-20ng of the T vector and use 1:1 or 1:3 ratio of PCR product for ligation.

usually u would have a manual with the T vector.

Promega has pGEM-T vector which does give u an idea abt such cloning. Check website for more info.

-scolix-

I have no pGEM T vector....so no manual ...... will be preparing the T vector...wud like more info on this....

-chimmi-

QUOTE (chimmi @ Aug 22 2006, 09:10 AM)

I have no pGEM T vector....so no manual ...... will be preparing the T vector...wud like more info on this....

If u dont have the vector I would suggest to PCR amplify with specific sites and digest the product and insert in vector of choice (of course with the respective sites) and finally sequence it.

-scolix-