A web forum for questions and answers (hopefully!) regarding the Suicide Polymerase Endonuclease Restriction (SuPER) methodology.

Monday, March 06, 2006

Questions regarding template addition and cleanup

QUESTION: I would like to know how much DNA templates should be added to the SuPER reaction mixtures and how much dna templates do you use after the SuPer reaction to perform the normal PCR.

First, there is no specific amount of DNA you should add to the SuPER reaction, and it may take some optimization to get the right amount. We managed well with something like 10-40 ng of genomic DNA in the reactions (25 ul volume) to remove plastids. However, the reaction could probably handle more genomic DNA. At some you will probably overload the reaction and get incomplete digestion. Thus, I recommend you try a range of different amounts of DNA until you find an optimum for your samples. Also, remember that the amount of genomic DNA you add isn’t really the relevant issue, but rather amount of target DNA you’ve added to the system. This gets us to your second question, which I’m afraid to say will also require optimization. The more dominant the template you are trying to remove, the less uncut DNA will remain after the SuPER reaction. If your system is 99% DNA to get cut with the SuPER reaction and you add 100 ng to the reaction, you will only have 1 ng of uncut DNA in 25 ul after the reaction. In such cases I often had to reduce the annealing temperature of the subsequent PCR reaction while keeping magnesium concentrations at 4.0 mM to get reasonable PCR amplification. However, if your target DNA to get cut is 50% of the DNA, you will have 50 ng remaining which will be much easier to amplify.

QUESTION: The second question is why don't you clean you DNA template after the SuPER reaction to avoid inhibition problems in the normal PCR ?

We wanted to minimize the number of steps involved in the reaction, and cleaning up of the DNA inevitably results in a certain loss of DNA. For those reactions in which DNA concentrations are really low after the SuPER reaction I was worried about the inability to amplify the DNA afterwards. Plus, I found that if I reduced the primer concentration and digested the enzyme, I could directly PCR the resulting DNA. You are free to clean up the DNA… there is no theoretical problem with it. If you can get enough DNA (perhaps combining multiple tubes) it probably will be even better.