TOPO TA ligation problems - (Jul/05/2013 )
I have been trying to ligate my PCR products into the Topo TA vector from invitrogen. I had success the very first time I did the ligation with a fresh PCR product (Not gel purified), but when I repeated the ligation with products extracted from a gel there was no inserts, I tried an A -tailing reaction with no success.
I am getting hundreds of transformants on Lb ampicilin plates but upon analysis with both PCR and restriction digest the plasmids appear to be closing without inserts.. HOW IS THIS POSSIBLE??? It is also reported in this paper http://aem.asm.org/c.../74/5/1649.full that as many as 34% of their transformants were closed plasmids.
So I have two main questions,
1.Does topoisomerase mediated ligation require presence of a buffer like those used in normal PCR to work efficiently?
2.Is there any explanation why my plasmids closing when they have a T over hang on both ends
Also, I am using MyTaq from bioline
Any advice would be greatly appreciated!
This second product, is it the same or different sequence from the fresh PCR one?
I think TA cloning manual doesn't say anything about PCR buffer requirement and if I recall right, I did successfull transformation with gel-extracted PCR products. They just need to be fresh too, i.e. not frozen for longer time, since they tend to lose A overhangs. What do you use for gel extraction?
TA cloning is supposed to be very specific, but sometimes the sequence in question is damaging for the bacteria so they cut it out somehow. I would personally say the empty vector alone would not recircularize that much, but more likely the ligation occurs and then it's cut off. You can test self-ligation on a separate plate without insert, I think it's even recommended in the kit.
What concentration of Amp are you using?
Thanks for the quick response!
I am actually using the kit to make a lone-linker DNA library, so I wanted insert seizes of around 2000bp but the first time I was getting an average of 1100bp using fresh PCR product (by PCR and digestion). So I repeated the PCR and ran it on a 1% agarose gel then extracted the region between 2000-6000bp with the QIAquick gel extraction kit, I tried to only expose the gel to UV for a couple of seconds to cut out the region, I eluted the DNA from the column with nuclease free water.
Regarding the closed vectors, I did a digest and the control tube (un-digested) appeared to be supercoiled from size comparison to the digest. I was reluctant to try the self-ligation test because of the kits cost, my supervisor might kill me! But I guess if its not working its the only thing I can do. I am using 100ug/mL ampicillin.
So when you just transformed the PCR product right away, you got only short fragments, so you wanted to enrich the higher-length fraction by isolating it from gel. What do you actually see on a gel within your region, are there visible bands or thick smear? Because gel extraction even using Qiagen kits lowers amount of isolated DNA considerably, and QIAquick has a high elution volume too so the product is low concentration. Also cutting large gel fragment reduces efficiency of gel extraction.
Did you try to measure the concentration of DNA in the isolate or run it on gel if it's visible? I'm afraid your main problem could be insuficient insert amount in your ligation. TA kits are designed for PCR cloning, therefore maybe expect higher amount of insert for ligation. Also 2000 - 6000 bp is quite a long fragment to ligate, it's not usual length of PCR product. You have to consider also that the bigger fragments would have much lower ligation efficiency so that would in principle bias the library towards smaller inserts.
Unless you are sure you have a plenty of isolated DNA, I would:
- try a MinElute Qiagen kit, that has reduced elution volume and thus higher concentration of final isolate
- try to improve efficiency of your lone linker PCR to get more product to cut
- enrich the 2000-6000 bp fraction by other methods if possible like I don't know.. try to use the gel extracted portion in another round of PCR (DNA amount may be sufficient as a PCR template but not as a ligation template)
I think u got the colonies because your agar had problem with antibiotics. Maybe they were old over than 1 month. Try to use the new agar plates with new antibiotics.
I usually use EX Taq Pol from Takara to get the product with poly-A overhang and then purify the products by gel. It works not bad.
Since the colonies have closed vectors (presumably with the antibiotic resistance gene), this is unlikely to be the problem.
I measured the gel extract output with a nanodrop and it came out as ~10ng/ul. I ran it on a gel and it looked fine but I guess it could be the number of inserts with A overhangs intact that was the problem.
I have just used the gel extraction products as template for PCR (which worked) and I've just and transformed the cells with the cloning reaction (which hopefully will contain my inserts!) I'll be doing restriction digests tomorrow, so I will post the results.
And yes, I don't think it is an antibiotic problem, the clones I tested all contained closed vectors with the antibiotic resistance present.