purification、ligation and transformation problems - (Mar/16/2008 )

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 DNA purification kit or promega DNA purification kit. After purification, I can see the DNA lane clearly from DNA gel. Then I do the ligation with the different molar 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 x 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 70degreeC 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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how do u cut your band from the gel? how long do you expose it to UV? I had problems when I expose my gel to UV for more than 10 seconds.
try ligation over night at 16C

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Hi,
I don't get you on this?
Do you run positive and negative controls along with your samples?
Thanks, best luck.

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hi,
are you sure you have used the correct restriction digestion sites. not to interupt with slection?

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Are you treating your cut vector with alkakine phosphatase?

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I cut my band from UV. The time exposed to UV i think is more than 10 seconds. I didn't know the time expored to UV was important before. I am not sure if it is the only problem with my ligation. I tried many times at 16C overnight. thanks a lot!

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No. i didn't do this!

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could you tell me how to run positive and negative control with my sample? I just do the positive control and negative control with my ligation and transformation to corform my ligase and compent cell works well. Thanks for you advice! expect your answer!

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That could be one reason why you aren't getting any positive clones - your vector may be ligating back together.

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I spent almost 4 months trying to clone a fusion gene and always ended up with either no clones or few clones missing alot of my gene. But when I minimized UV exposure, Screening was almost 100% positive with realy high transformation efficiency.
According to your controls, it seems to me purification problem as you said.
In control number 1 you are not supposed to get anything and that's fine
control 2 worked because there is no gel purification
control 3 showed nothing because of the purification.

Try what I said, just cut the bands as fast as you can from the gel. No need to cut the band exactly, little bit of gel won't affect your purification. I used Qiagen in my purification and I think promega is good enough.
Give it a shot and let me know how it goes.
UV affects the ends of your fragments that'swhy they might not ligat.

Good luck

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