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Cloning Problem! I need help

pGem Vector PCR colony ligation reacton

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#1 RaquelCB86

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Posted 12 October 2012 - 08:55 AM

Hello Everyone!

I'm a younger researcher and I'm doing cloning for my first time and it doesn't work so well. I'm having many problems in differents step of cloning and i need help to understand the reason for that.

I did a PCR to amplify my DNA fragment of 422 bp before cloning and it had a big quantity of primer-dimers. I have excised the PCR product of interest to cloning. But, i had a lot of problems. I used the pGem-T vector system. First, when i studied 4 samples, i've had colonies white and blue (but not more than 80 colonies) mostly in 3 samples. So, i thought that it was a ligation reaction problem. i've tried ligation reaction overnight at 4ºC. Ok, this time i had colonies (not more than 20 white colonies) in all samples but no more than 100 colonies as theorically expected. I selected 4 white colonies of each sample and did a PCR colony. In the gel agarose, in a total of 16 reactions i have only had the vector+insert in two (of the same sample). The other reactions shows a lenght bigger than the vector but smaller than the vector+insert. So, my doubt is what is happening in this all process? something is failing. I'm afraid that the reason of the cloning process isn't work is about some inhibitor as pyrimidine dimers. But, after i've purified the PCR product i ran a gel and i saw a band (not so strong) but a clean one. After purified the PCR product i can see the primers-dimers or not?

Thank you so much for your attention and please help me!! I want a lot to understand where i'm failing!

Best Regards,

#2 phage434

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Posted 12 October 2012 - 01:24 PM

Gel cleanup does not completely remove small PCR products. And these products are often hard to see on a gel, since the brightness is related to the mass, not the molarity of the DNA fragments. You can have many many small fragments which don't show up on a gel, but will show up in cloning. I'd recommend trying hard to optimize your PCR reaction conditions to reduce the primer-dimer formation. Often this will require a redesign of the primers.

#3 RaquelCB86

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Posted 14 October 2012 - 01:00 PM

Thank you for your answer! But do you think that my cloning problems can be due to the quality of DNA fragment?

#4 phage434

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Posted 14 October 2012 - 01:43 PM

Almost all "ligation" problems are either poor quality DNA going into the ligation, or poor transformation efficiency of the competent cells. Ligation almost always just works -- one potential issue is the stability of the ATP in the ligation buffer. In your case, I believe the problem is likely the poor specificity of your PCR reaction in producing the product you want rather than priimer-dimers.




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