TOPO cloning - Too much background (Oct/04/2007 )

I am cloning PCR products into TOPO vectors and I am having a background problem. I get background colonies just by adding the TOPO vector + salt soln and dH20. I have done the proper controls with the cells and no DNA so its not the cells. The kit is brand new so I cannot imagine the vector is contaminated. The background is not THAT high but it is there.

A bigger problem I am having is with the background of another control. I am trying to shotgun clone short pieces of DNA for sequencing purposes and the protocol is to digest the DNA (human genomic) with MseI then ligate on linkers (also digested with MseI). An initial PCR reaction is done using primers specific to the linkers, then a nested reaction, was performed again with primers to the linkers...afterwards the products are immediatly cloned without purification into the TOPO vetor. Using genomic DNA with no linkers I get low background as expected since there was no amplification (same as with no DNA), but my linker control (PCR performed without DNA, just with linkers) yeilds a massive amount of colonies. If the linkers are being extened by the polymerase into short dsDNA capable of being ligated into the vector...then I dont understand how this protocol would work at all...but it is a well established protocol.

...am I missing something?....how do I get rid of that background?

-A1000miles-

Hi A1000miles,

I'm sorry, that I don't have any suggestion, but I'm having a similiar problem: I haven't done negative controls, but after my last few TOPO cloning reactions I got a vast amount of colonies, while most of them where negative (no contamination just TOPO vector without any insert). I've done HUNDREDS of mini preps
Luckily at least some where positive...
My PCR fragment (absolutely freshly used) is about 400bp long. Before TOPO cloning I never do any purification either. I also tried a fresh kit.

Hopefully, someone comes up with an idea...

best wishes
asile

Hi, this is my first post so be kind!

Just trying to run through things logically...is the following correct regarding your protocol?
Your protocol involves digesting genomic DNA with MseI, ligating linkers with compatible MseI ends, and your primers bind the linker cassette and your known genomic sequence to amplify an unknown junction fragment?

I am also using a cloning technique that utilises linker cassettes. To make mine, I order the primers with the restriction enzyme overhangs. I get them HPLC purified...this is more expensive but you know exactly what your working with. The 2 primers are annealed and you end up with a linker cassette with compatible restriction enzyme overhangs. Perhaps this is why you are getting amplification of your linkers during PCR.

Did you get white colonies with the vector, salt solution and dH2O only (aka contamination)? I have also just tried out the TOPO TA kit, with the Mach1 cells and had good success. I only used 20 ul of cells (instead of the recommended 50 ul) and this gave me 20 - 70 nice big colonies....mostly white. You could try using less cells? The fragments I used had been gel purified. My fragments were quite small - between 100 and 330 bp. Maybe you could gel purifiy your random fragments...you would need to cut out a large slice of gel that contains the band sizes you are after? This should exculde any primer dimers being put into your cloning reaction (since smaller fragements seem to clone easily).

Hope this helps!

Just trying to run through things logically...is the following correct regarding your protocol?
Your protocol involves digesting genomic DNA with MseI, ligating linkers with compatible MseI ends, and your primers bind the linker cassette and your known genomic sequence to amplify an unknown junction fragment?

yes, exactly

I am also using a cloning technique that utilises linker cassettes. To make mine, I order the primers with the restriction enzyme overhangs. I get them HPLC purified...this is more expensive but you know exactly what your working with. The 2 primers are annealed and you end up with a linker cassette with compatible restriction enzyme overhangs. Perhaps this is why you are getting amplification of your linkers during PCR.


The linker oligos I order, one is a regular oligo, and the other is modified with Phosphate on the 5' end and an amino group on the 3' end. This was published in Wu et. al., 2003 Science, volume 300, pg 1749. The modifications on the linker SHOULD prevent amplification of the linkers with the initial primers and the nested primers.

Did you get white colonies with the vector, salt solution and dH2O only (aka contamination)? I have also just tried out the TOPO TA kit, with the Mach1 cells and had good success. I only used 20 ul of cells (instead of the recommended 50 ul) and this gave me 20 - 70 nice big colonies....mostly white. You could try using less cells? The fragments I used had been gel purified. My fragments were quite small - between 100 and 330 bp. Maybe you could gel purifiy your random fragments...you would need to cut out a large slice of gel that contains the band sizes you are after? This should exculde any primer dimers being put into your cloning reaction (since smaller fragements seem to clone easily).

I did get some background with just the TOPO TA vector alone, but I get no background with no vector at all. The TOPO TA vector control doesn't give TOO many colonies that it is a major problem...my major problem is with the linker control. I ran the PCR product (what I am cloning) through a Qiagen PCR purification spin column to try and get rid of <100bp DNA even though some products below that limit could potentially provide postive results. It did reduce my background somewhat, but it is still a problem. Right now it looks like ~40% of my colonies are background.

Hope this helps!
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Yes, I have read Wu et al. (2003). Are you trying to do LM-PCR?? I am working on a LAM-PCR assay, which is similar but does have fundamental differences.

Sounds like your linkers are giving you a lot of problems. You could try gel extracting your bands instead of the column? I guess you are getting many bands on your gel so you might have to cut out the bands in groups.

Maybe its worth designing your own linker? I was having a lot of problems the LAM-PCR protocol. One of the main problems turned out the be the published primer sequences. Once I designed my own primers, the assay started working perfectly! Alternatively, in the LAM-PCR we use a high concentration of linker to drive ligation, maybe you could try different concentrations to reduce background?

Sorry I can't be of more help!

You could get better at throwing away the empty vectors. Try colony pcr rather than minipreps -- it's a lot faster for large numbers of samples. I don't know if it is relevant to your project, but you could also know the size of the insert from this.