TA cloning - (Feb/16/2005 )

hi,
I am trying to clone two genes in pQE30 UA cloning vector.
I am not getting clones with the insert.
1.Could it be that vector or the 2x ligation mix has been degraded because of repeated freeze-thawing?
2.My pcr product may not have 3'-A residues?

How do I add 3'A residues to the pcr product , instead of buying qiagen A-Addition kit?
Could you give me detailed protocol?

I have tried running the o/n ligation (vector+insert) on the gel. Vector is 3.5 kb, and my inserts are of 800bp and 1.2kb. On the gel I can see dense pcr products, and at 4kb is my vector, and there are faint bands of approximately 5.5 kb and 6 kb respectively.
But when I run the gel for a longer time, those faint bands disappear.
I dont know if those are the right ones.

Anyway, I have done the transformation.
Could someone help me what else I should be doing if it doesn't work?

thanks.
shilpa.

Hi,
I am a newbie to PCR. I would like to tell you that, If u r using Taq polymerase, ur PCR product will have A overhangs because Taq will add A to the ends of your PCR product.

I have never tried to add overhangs to my PCR product. I made a search on google for this and found one page that u would be interested in:

http://www.eng.buffalo.edu/~coffroth/ssr-day3.html

Devender

One more method to add A overhangs>
See Chapter 2 on :
http://www.promega.com/guides/subcloning_guide/default.htm

1. about A overhang : i also think you should definitely get it with taq, if you are using a different polymerase check the product specifications.
there are rumors that PCR products which have been kept long, freeze/thawn ,etc., may lose their overhangs - i don't know if its true but would try fresh PCR product just to be sure. also be sure to clean product before ligation
2.if you can get your hands on a PCR product which has recently been used as insert and worked - ask around - that would be really good as a positive control for vector, buffer, etc. also - is the transformation working well with other plasmids?
3. about the dissapperaing bands - the longer you run your gel , the more diffused the bands get. so faint bands can dissappear, thats normal.
about the sizes - closed plasmids migrate differently from linear form so its hard to know if they are the right ones. but i think thats encouraging.

Good Luck!

The other posters are correct. If you use Taq polymerase, you will always have an A overhang. Did your clone kit come with a control insert? Try using that to help isolate the problem.

Freeze-thawing does degrade the buffer. Also make sure that the buffer is completely brought back into solution. If you noticed, there are always white precipitates in the buffer. That's ATP and it is required for the ligation to occur.