ligation and transformation problems! Urgent!
chunmei zhuang
Sunday, 16 March 2008 22:29 UTC
Hi,
I am trying to ligate my gene 1400bp to a 6.4kb vector. I double digest the insert and vector from two plasmid with bglII and xho1. After that I purify the insert and vector for the gel with bio-rad kit or promega kit. Then I do the ligation with the different ratio insert: vector=3:1 or 5:1. The T4 DNA ligase I use is from invitrogen. The protocol for the ligation is room temperature 4h or 16 degree C or 4 degree C overnight. The problem is that I can get nothing after transformation. I tried many times, but still get nothing. I am nearly crazy now.
I did the positive control with PUC19. The efficiency of my competent cell from invitrogen (Subcloning Efficiency™ DH5α™ Competent Cells) is no problem. The transformation efficiency is 2 × 10E7 at least.
I did another test with the PBSK plasmid. Cut a single site with BamH1, and then I did three transformations with:
1. 1ul Digest solution
2. 1ul Digest solution(after inactive enzyme at 70 10min) add ligase at room temperature 3h.
3. After purification, 1ul product (DNA gel check the lane very well) add the ligase the same with 2).
The result was I can get nothing from 1) and 3), for the 2) I can get many many clones. The problem seems like is the purification problem, but I don’t know what the exactly problem is. I have used three different purification kits from different company. The 50xTAE stock solution and agarose I used is from fisher company.
Could anybody give me some suggestions? I would be very much appreciated if you could answer my questions!
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Replies
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Hi Chunmei,
It might be worth checking the wavelength of your transilluminator. It’s important to only use long wave UV to visualise the DNA in the gels and to expose the DNA to the UV for as little time as possible. Short wave UV is extremely damaging to DNA and can dramatically reduce cloning efficiencies, especially of DNA fragments with ‘sticky’ ends.
Hope this helps and good luck with your cloning!
Best,
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Hi dorothy,
I appreciate you very much. I will follow your advice and try again. I never notice expose DNA too long will damage my DNA before. I think your advice is quite valuable to me. Thanks a lot! -
You can dephosphorylate to prevent the vector from self-ligating as well. I am having a lot of problems with a ligation right now myself, I can sympathize!!
I gel purify my stuff with a Qiagen kit, I have never used anything else.
Check the concentrations as well, they might be too high, ligation concentrations can go extremely low, down to pico and femtomolar.
If the T4 is going through repeated freeze-thaw cycles is may be degraded by now. It is helpful to aliquot T4.
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