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Transformation problem. Please help!


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#16 phage434

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Posted 18 August 2009 - 08:55 AM

Some comments:

* I assume you have checked that there is sufficient 5' overhang on your PCR primers to allow the DNA to be cut with your enzymes. Bases in your primer are cheap.

* That's a lot of template for a PCR reaction. With 1 ug of template DNA, your amplified product will only be a few times more concentrated than the template DNA. Try to amplify with much less template to reduce the junk DNA in your final reaction.

* The DNA concentration in your restriction digests is way too high. Typically you want 1 ug in a 50 ul volume. You have 5 ug in a 30 ul volume. Also, I would not be using that much enzyme. Try 1 ul. Overnight is unnecessary and possibly damaging to your DNA. 1 ul of enzyme will have 20 units or so, enough to cut the 1 ug of DNA you should be using in your reaction in less than an hour at 37C. Heat kill for 20 minutes at 80C. You can conveniently do both in a PCR cycler.

At this point, I would go directly to a ligation. Too much handling of DNA is difficult and often damaging. If you have a good PCR band with few side products, and good digestion of your backbone, this ligation should be easy. As memo suggested, control digestions of your vector with each enzyme will make sure things are cutting. Use the gel to evaluate relative amounts of insert/vector. Remember that the ideal spot is 1-3x as many molecules of insert as vector (molar ratio), but people tend to worry too much about this.

The blunt ligation is much more difficult than a cohesive end ligation. I would probably try it with a PEG containing buffer ("quick ligase.") What I would really do is avoid having to do a blunt ligation. Overnight, as suggested, at low temperature. You don't mention the amounts of DNA in your ligation. You are probably using too much DNA. I would try 20 ng of vector and the correct amount of insert by molar ratio, likely around 10-20 ng.

You say very little about your transformation protocol. In particular, you should add a maximum of 2 ul of ligation mix to the competent cells. If you are adding 20 ul of ligation product to 50 ul of cells, you will have very low efficiency transformation. I STRONGLY suggest doing a control transformation with 10 pg of pUC19 DNA (or some other convenient plasmid) to establish the competency of your cells.

#17 planktonica

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Posted 08 September 2009 - 09:15 AM

If you are adding 20 ul of ligation product to 50 ul of cells, you will have very low efficiency transformation. I STRONGLY suggest doing a control transformation with 10 pg of pUC19 DNA (or some other convenient plasmid) to establish the competency of your cells.


Hi Phage, I am officially a fan of yours :)

I just have a question regarding your post. I usually add little amount of ligation product to my transformation, but I do it because I was told so. Nobody has NEVER explained to me why adding 10 uL or more of ligate will result in a low efficiency transformation. Could you explain me that?

Thanks

#18 phage434

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Posted 09 September 2009 - 04:23 AM

As with many other things having to do with transformation, the root cause of this is obscure. Presumably the addition of low concentration solution to the high osmolarity transformation buffer results in change in the salt concentration of the solution suspending the cells. Given their fragile condition, this may be enough to kill a substantial number of them, or to upset the dynamics of the transformation reaction. Short answer -- I don't think anyone knows. I once tried to formulate a 2x concentrated version of transformation buffer which could be used to dilute ligation product to the same salt levels as the competent cells. I had mixed success in using it, probably because of bad control over the concentrations and the pragmatic difficulties of doing large numbers of experiments with many many variables.




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