Too many negative clones using Directional TOPO Cloning - (May/23/2013 )

Hi everyone,
I have been working on this project for months now and have litterally done hundreds of minipreps searching for a positive clone. I am using a Champion pET Directional TOPO Expression Kit. So far I have only gotten two positive clones out of the hundreds that I have checked and I am not sure why the efficiency of the rxn is so low. I always get about a hundred clones per LB/AMP plate. I usually do the whole reaction in one day where do PCR to amplify the insert, carry out a gel extraction (using a Qiagen kit), run the ligation reaction and then transform the bacteria and incubate the plate at 37 degrees celcius over night. Then I carry out a miniprep and run a PCR to search for positive clones. So when I run the PCR I always see the backbone of the plasmid which is about 240 base pairs in the negative clones so I know that the minipreps are working properly. By the way my inserts are 600 bp and 1500 bp long so in general the length of the insert should not be a problem. When I ran the same reaction with the control insert I was able to get one positive clone out of ten, which is no where close to the 90% efficiency that the kit claims. Also I have tried this same reaction with a newer kit and it still did not work. By the way I also called invitrogen tech support and it was their beleif that the plasmid was bad which is why we got a new kit. I should also note that I followed the directions for the ligation rxn without any deviation and used the proper amount of DNA for the rxn.

So all in all, I know that the minipreps, transformations and (most likely) the kit is good. So why am I getting so many negative clones? Are there any alternatives to the invitrogen kit that will give me less background? What could I possibly be doing wrong during the ligation reaction that is implied?

Sorry for the long post I just wanted to be as thorough as possible. Any suggestions will be greatly appreciated.

One question I have is how you are quantifying the insert amount. Perhaps your insert DNA is present at much lower levels than you think. Perhaps your DNA is being affected by UV exposure on the gel. I would try your reaction without gel purification, simply cleaning up the PCR product (column, exo-sap, or ampure beads).

Idea: after you do the transfomration, let the transformed E.Coli sit in the fridge overnight, or more, and then plate the following day. What I find is that greatly increases the frequency of positive clones. With one recent ligation I checked a 100 clones and found nothing, but when I let the transformation (after shaking) sit in the fridge for 6 days before I plated I got several positive clones. This was a total surprise to me but it worked. Go figure.

So, I am quantifying the insert using a nanodrop 2200, which is supposed to be pretty accurate. It is definitely possible that my DNA is being affected by UV exposure since I use a gel imager to take a picture and then use a gel dock to excise the gel slice or interest.

By the way srpres when you say to let the E. Coli sit in the fridge overnight and then plate them do you mean to let them sit at -20 or -80 degrees celcius?

4 degrees (refrigerator)

Well, you are not alone and you don't need to blame yourself. TOPO cloning is known to have backgroun problem. Vectors prepared according to Invitrogen's protocol suffers from batch to batch variations. If you tell their tech support your problem they will send you a new kit at no cost. They knew their problems well.

It happens from time to time. The background issue is most obvious when you don't include any inserts in the reaction. My most recent TOPO cloning with 6 different fragments ranging from 400bp-1kb results in "0" rcombinant. I mini preped 4 from each plate using spin column based method (equivalent to Qiagen's miniprep but with silica membrane spin columns bulk ordered at much better price from Epoch Life Science) and sequenced all 24 plasmids. The are all backgrounds with slightly different sequnce. I pulled out Invitrogen's genome research paper, the original paper on Invitrogen's topo cloning vector and all Shuman's original research paper on topoisomerase I and TOPO cloning. Together with the sequencing data I believe all the background came from various steps during the TOPO vector preparation, the digestion step, the adaptor ligation step etc.

Since then I have tried to make my own topo cloning kit by eliminating some of the complicated steps in the original method. The best 0 background method that I found is what I called "Reverse Topo Cloning". For this method you simply add topoisomerase I recongnition sequence(-ccctt-) to the ends of your PCR primers. Nicking the PCR product with vaccinia topoisomerase I will leave two sticky ends together with the enzyme covalently linked to the T residue. If you design the primers in such a way that the sticky ends matchs restriction enzyme digested vectors then you can easily ligate the TOPO treated PCR fragment in to the digested vector at 100% efficiency without any backgound issue. Just be reminded that the vector has to be dephosphorylated first!

I have also tried to make the original TOPO cloning vector and found the same background issue although not as bad as some of you experienced. I am still in the process of improving and perhaps completely eliminating the background issue. I certainly will be happy to update you once I have good results. For the moment I am only using homologous recombination based method (SLIC or Gibson assembly).