HELP! cloning problem with pET22b using NdeI and XhoI sites - (Mar/23/2011 )

Hi everyone,

I'm currently working on cloning of my insert, about 700bp, into pET22b vector using NdeI and XhoI sites.

The problem I'm facing is that, after ligation of my insert to the cut vector and performing a colony-PCR to screen for positive clones with the recombinant construct, I did see bands corresponding to my insert (700bp).

However, when i send my construct DNA for sequencing verification, it turned out to contain the vector sequences only.

Then I tried re-streaking the positive clones onto a new plate (Perhaps there might be a mixture of cells: those with and without the insert). And did a colony-PCR on the restreaked clones again. I picked 8 clones and all 8 contains the insert.

I did a plasmidprep of the recombinant construct, followed by a restriction digestion using the 2 enzymes, NdeI and XhoI. But I did not see any band for the insert that should have been cut from the recombinant construct.

I am just wondering how I could get insert band in colony-PCR but negative results for sequencing verification.

Anyone encounter similar situations? Please help me because I've been doing this for the past two months.

Will definitely appreciate any help rendered. thanks in advance!

Have you done a no-template control on your colony pcr reaction? My guess is that one of your pcr reagents is contaminated with template. Or you are contaminating your pcr tubes/tips with template from e.g. your autoclave. Colony pcr is a quick, but quite inaccurate method.

phage434 on Wed Mar 23 12:37:04 2011 said:

Hi,

thank you for your reply. Really appreciated it.

I've performed colony-pcr together with a negative control (pET22b only) and a positive control (insert). I see bands corresponding to each respective control.

The problem now is that I can see my insert band when I performed colony-pcr, but yet when I send for sequencing verification, it turn out to be the vector only (without the insert ligated into the vector).

I'm currently trying to restreak my clones, up to the point where I can purely get recombinant clones only. I was suspecting that the clone might be of a mixture at the beginning: some with wild type pET22b, some with pET22b-insert construct.

whats your opinion on this?

thanks!!

Could be. Or you could be seeing DNA left over from your transformation on the plate. Is one of your primers on the pET vector and the other on your insert? That reduces problems quite a bit.

phage434 on Wed Mar 23 15:29:47 2011 said:

Thank you for your reply!
my insert doesnt have the restriction sites, so I design primers containing NdeI and XhoI to amplify my insert (so that my amplified product contains the restriction sites).

The primers I'm using for colony-PCR is T7 promoter primer (Forward) and XhoI site-containing primer (Reverse).

Currently, I'm sending those restreaked clones positive for insert in colony-PCR for sequencing verification.

If results turn out to be negative again, I'll try sequential digestion, first with NdeI, followed by XhoI.

what's your take on this?

thanks again!