Hi Niti,

If you are amplifying the gene using Taq polymerase then pGEM-T vector is the best to get clone. Our lab mainly work on metagenomics and clone library. We generally use dream-taq pol and transform in pGEM-T vector because its transformation efficiency is so high. Hope it will help you..

https://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/0/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Protocol.pdf

Best of Luck!!

Try pGEM-T or pTOPO vectors.

Hi Niti,

If you are amplifying the gene using Taq polymerase then pGEM-T vector is the best to get clone. Our lab mainly work on metagenomics and clone library. We generally use dream-taq pol and transform in pGEM-T vector because its transformation efficiency is so high. Hope it will help you..

https://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/0/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Protocol.pdf

Best of Luck!!

Thank u Debaprasad,

Is pGEM-T an expression vector ?

can i carry out sds page and enzyme assay aafter obtaining a positive cclone?

No, its not an expression vector, its only a cloning vector. For expression vector I'll suggest you BL21-de3 vector. And another important thing, if you are going to check expression then try to avoid pGEM-T. If you read the mechanism of cloning in pGEM-T vector, you will find there is a A-T overhanging in the vector to which the overhanging of PCR product bind. And this overhang can create a problem during expression of the gene as the basepair numbers will be higher for AT tail which hamper the ribosome binding as well as promoter finding.

I'm using pET 28a as cloning vector and again transform the positive clones in BL21-de3 expression vector. Then inoculate the colonies in LB and induced with IPTG when the O.D. reach 0.8. Then only you can check in SDS-PAGE whether those colonies give your desired protein or not. If you need full protocol let me know.

The above was a great help, it cleared my doubts.....if you could lend me detailed protocol it would be great for me as a beginner in the task.....

Choice of expression vector depends on which expression host u want use. If u want to express it in Prokaryote like ecoli then select ecoli BL21 as expression host and vectors from pet systems . Similarly for yeast expression use yeast expression vectors eg. pYes

Hi, I used pRSET expression vector from Invitrogen for my cloning and E. coli BL21 for protein expression. It works for me and the recombinant protein obtained has the activity (ferroxidase activity) I was looking for. Is important to know which version of the vector is better for you cloning strategy, also you will have to optimize you expression protocol, etc. You can use SDS-PAGE and Wb to verify your expression, protein product, etc. If you need more detail let me know.

I highly recommend invitrogen TOPO cloning kit. The best feature is "NO ligase" is necessary and it only involves 5 mins incubation with the vector.

pGEM-T vector

I have got good results with the Champion™ pET Directional TOPO®

Expression Kit

http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf

This one contains the pTOPO for cloning and BL21 Star™(DE3) cells for the subsequent expression

http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf

Thank u Susana .!

I also got good results from Champion™ pET Directional TOPO® Expression Kits. If you just need some protein from this geen, you can express the in vitro. In vitro expression systems use an efficient coupled transcription and translation reaction to produce high yields of full-length, functional protein from E. coli cell lysates. The time-consuming steps of cell-based protein production have been eliminated; now, in only two hours, you can produce protein starting from plasmid or linear DNA.