Making more empty vector - (Feb/08/2007 )

Hi.

I'm just running out of my batch of pTarget bought from Promega.

I was wondering how to get more by myself. Do I just ligate without any insert, put it into a plate without antibiotic, take one colony and do mini/maxiprep? Is that all?

I did in the past but in my cleverness I put it into a plate with antibiotic and I think now all my new obtained plasmid is resitant to ampicillin regardless got the insert or not.

Can anyone tell me how to do this? What can it go wrong? What are the main disadvantages vs buying from the company?

I would appreciate it if you can give suggestion or share your own experiences.Thanks.

I guess pTARGET is a T-vector. so i am sorry but you cannot just ligate the linear vector. It has T overhangs at both ends. so ligation would be impossible. you need to buy one, I think.

How do the ends of this vector look like?

In one old post pernesblue had made good drawing about a linker for a similar problem.

http://www.protocol-online.org/forums/inde...26&hl=bsmbi

check the last posts.

u can actually fill in the overhangs by using polymerase. then ligate and transform. this will help you getting more of your pTarget. but it will no more be a T vector.

well, you could digest with EcoR1, then ligate at a low concentration, which would give you the recircularised vector. But as someone already pointed out, it's no longer a T-vector, which would defeat the purpose if you want to use it for an easy cloning vector. You can make it into a T-vector again, but it's fairly fiddly. Plus, the vector is usually provided at a low concentration (about 10 pg/uL), so manipulating it will be a bit hard.
A better way to save $$ is to do half or even quarter reactions - eg, if the protocol calls for 1uL vector, 2uL PCR product etc, use 0.5 of vector and 1 of PCR product. Or even less, I have gone down as low as 0.2uL of vector, and it still worked (this is not for your vector, but for a similiarly-designed TA vector).

Hi,

You can generate a PCR product as described in this post with Ahd I sites:
http://www.protocol-online.org/forums/inde...0686&hl=ahd

then T/A clone the PCR product or via RE ligation into the pTARGET. If you want a T/A cloning vector, just digest with the RE, as described in the above post.

Thanks for all replies. I think I go for the easy way and I will dilute my ligation reaction to half to begin with and hopefully it will work. I thought of doing that in the past but I didn't dare to do it. Now I will.