Help with ligation using Topo TA - (Oct/27/2006 )

The Problem: I have no (or occasionally 1 blue) colonies with transformation.

Background: Transformation control works, using pUC19. Following invitrogen protocols exactly. Have vector:insert ration at 1:3. AMP at 100mg/ml. XGAL used. Ligation using 4kb pCR 2.1 at 14 degrees overnight. Insert is 280bp. Kit is new and stored appropriatly. Reqular TAQ used, final extension of 7 minutes. no gel purification... I have a single strong band visualized after PCR. Am diluting PCR with sterile distilled water to get right vector:insert ratio.

Other info: Primer has a 5' T... Could this be the problem, limiting A placement by TAQ??

Thanks- Matt

Hi,

I'm not sure, but as I remember, the advantage of the Invitrogen Topo cloning Kit is that you don't need to ligate over night. You just mix purified PCR Product and Topo vector incubate for a couple of minutes and that's it. It's special vector mix, where the ligase is included already.
Additionally, you should not need any ligation control (espeaccially not with another vector) as the insertion of your gene cuts a gene which codes for a toxic product. So no ligation, cell dies due to the production of a toxic protein in the vector.
But may be I am talking about the wrong kit. Better check the supplier instruction. That might be the easiest.


dedee

As dedee says, the Topo TA cloning kit is not used with extra ligase. It should be fast and easy. That is what you are paying (a lot!) for. Read the instruction manual -- all that is required is to mix the PCR product with the vector, water, and salt solution, wait 5 minutes at room temperature, and transform cells (provided). I recommend cutting volumes in half to minimize costs, but you might not want to do this the first time.

If you are ligating and waiting overnight, you are reading the wrong protocol.

Right. You have the TA cloning kit, not the TOPO-TA cloning kit, as you originally said. There is a big difference in the protocol, and you are following the correct one, I believe. What competent cells are you using? When you do the pUC19 control, what transformation efficiency do you measure? When you add ligation mix to the competent cells, what volume of each do you use?

I am using the INValphaF'. Trans. eff. is within specs, about 1 x 10^9 cfu/ug. I am using 1/2 reactions, so 25uL of cells and 2uL of ligation mix. I'm quite sure that is is not the transformation step that is bad, as the pUC works as expected.

I read a paper last night about (Brownstein 1996) where he notes that TAQ has a hard time adding A tail when reverse primers with have T on the 5' end.. and imagine that- I have a T on my 5' end. I'm going to try primers that I know have cloned sucessfully before to make sure this is not a PCR/primer issue.

Ever hear of this?

I sympathize as I was once using an Invitrogen TOPO kit. It never worked for me, I personally never got a clone to work with Topoisomerase. I avoid them like the plague now. Others on this forum, or even in the lab upstairs say it is great and powerful, this has not been my experience. I would next time use a TA cloning kit that uses ligase instead of topoisomerase. To be fair there are those who like your kit though.

Neil Stewart