Clonning with one restriction enzyme - Some help please (Nov/17/2006 )

Hi

Im trying to introduce my insert into pTarget. I just want to chek if I'm doing things correctly because I didn't get any colonies in my plates:

- I used Sal I with my insert which cuts one end. I did PCR clean up.

- I used Sal I (3.5h 37C) with pTarget (1ug) which cuts into the restriction site zone. Then I used CIP with the product for 1 hour 37C. I run the sample on a gel, cut and purify it in order to eliminate CIP and the the restriction enzyme.

- I run a sample with both products getting a single band for each.

- Subsequently I did the ligation reaction as indicated by Promega, 60ng pTarget, 3:1 of my insert and T4 ligase, etc.

Do I have to do something with the other end of my insert? It's suppossed to have A added by the enzyme.

Do you think there is something wrong with this methodology? Is there something I'm forgeting?

Thanks.

PS: I must say that the pTarget vector used is not the original one, but one obtained by Miniprep from the original one.

Hi,

If I'm not mistaken, your vector is a TA vector? And you're doing the SalI digestion on the vector and also the insert (assuming that you purposely introduced SalI site on the reverse primer) to ensure unidirectional orientation of your insert in your pTarget?

Well.... it should work, but why not try doing TA cloning as well? Transform, pick random colonies, screen for correct orientation by doing RE with SalI. If RE product is single band at a size that is the sum of vector+insert, then it's your target clone. If there's two bands, then it's the reverse orientated clones.

Still... I think the latter is worth the try compared to the former method. Though T/A cloning sometimes tend to favour certain orientation, most of the time... they're just 50-50%. That'd be easy for you to screen hopefully .

Thanks for the reply. You are absolutely right. That's what I wanted to accomplish.

In fact, it would be my first time trying to do ligation through restriction digestion and I thought it was a good starting point.

About the TA clonning, it was my next step. I was planning of doing on Monday. The only thing is, will I ever learn how to do ligation with restriction digestion?

However, I would like to know if my method was right or wrong in theory.