5 replies to this topic

[joy123]

Hi, I plan to make a subgenomic library.

I am interested in a protein encoded by a 2kb gene from a bacteria, but I cannot find which gene it is (I only know the gene size). So I want to digest the bacteria genomic DNA with Sau 3AI, purify 2kb fragments, ligate to a vector, and express it in E.coli. Then I would use a selection method to find the E.coli that have the vecor with my interested insert.

Now I have problem in choosing the vector. Would you please give me some suggestions? I am new in this area and need your help. Thanks a lot!

[Koeng]

I am new too, but I would use either pUC18 or pUC19, because they give high yields of plasmids. Good luck with that!

-Koeng

[Koeng]

Also you can use them because of the "blue white" screen. The MCS is inside of the lacZ gene, so plasmids that where sucessfully ligated will be white, while the un-ligated ones will still be blue.

-Koeng

[zogene]

Hi

I would suggest to clone your gene to expression vector since you want to purify recombinant protein, pGEX and pET vectors are popular for this purpose, I did not do it by myself my boss did and I will begin with it within few days hopfully, do not forget to use expression bacteria either in your experiment, good luck.

[joy123]

zogene, on 09 July 2012 - 12:28 PM, said:

Hi

I would suggest to clone your gene to expression vector since you want to purify recombinant protein, pGEX and pET vectors are popular for this purpose, I did not do it by myself my boss did and I will begin with it within few days hopfully, do not forget to use expression bacteria either in your experiment, good luck.

How about pET28b, and BL21 (E.coli DE3)? Do you think it reasonable? Thanks!

[zogene]

Hello

yes I think they are fine, good luck

regards,
zogene