TA-cloning vector contamination - (Mar/27/2007 )
Hi,
I'm trying to clone a 5.3kb promoter fragment into the pGL3.basic reporter vector. Therefore I first cloned the PCR product into a TA-vector (pGEM-T easy, Promega), digested the vector with the two restriction enzymes I tailed my PCR primers with (MluI & XhoI) and gel isolated the fragement. I used this product for ligation with the digested pGL3 vector and transformed the ligation mixture afterwards.
However I only found a few colonies on my plate. After DNA isolation from those colonies and digestion, there only is a band at the size of 3kb which ist exactly the size of the pGEM-T vector (whereas the pGL3 vector ist about 5kb). I'm wondering if it is possible that I have a pGEM-T vector contamination in my ligation and if so, if the actual linearized vector would self-ligate and thus, be transformed?
Has anyone experienced similar problems?
Carolin
I checked it out for you and I don't think the pgem vector has an xho. so you're not getting a ligation product and are only getting self ligation from the single mlu cut.
www.promega.com/tbs/tm042/tm042.pdf
correct me if I'm wrong and this is not the right vector map.
in the future you can try the tech dev open science project for general molecular biology help. staffed by scientists to help with your experiments, even help with
www.promega.com/tbs/tm042/tm042.pdf
correct me if I'm wrong and this is not the right vector map.
in the future you can try the tech dev open science project for general molecular biology help. staffed by scientists to help with your experiments, even help with
Thanks for your reply!
However I don't quite get what you're saying. I know that the pGEM-T doesn't have a Xho. But since my PCR primers are tailed with a Mlu and a Xho site and then ligated into the pGEM-T by means of the A-overhang, I should be able to cut my PCR fragment out of the pGEM-T with a Xho/Mlu double digest, right? I isolated this fragment and used it for ligation with the Xho/Mlu-digested pGL3. With this transformation I'm having the problem that I only get colonies apparently holding the pGEM-T (at least the band after digesting with Mlu or Not shows a band at 3kb).
Sorry if I didn't understand you correctly but could you please be a little more precise? :-)
Thanks!
my fault, missed the TA part. Are you using a Taq polymerase or proofreading polymerase? Most pfu, proofreading enzymes will not leave an overhang for you, but Taq will. If using taq, you'll have to sequence your inserts to make sure there are now mutations, b/c it has less fidelity. But, if that's not it, maybe your plasmid is just old - went bad during storage...