Hi researchers,
I'm doing PCR from a 8kb fragment from genomic DNA. Since I need to do many samples, I find that the template quality seems affect the yield.
Can see the attached figure. Just focus 7-12 lane. Each two adjacent lanes are the same sample and the primer is the same. Everything in the PCR reaction system is the same, except for the template. I also try to use the same amount of template, 200ng for each sample. But the actual amount is lane7,8 200ng, lane 9,10 200ng, lane 11,12 170ng.
If the gel is like this, will it indicate the template quality for lane7-10 are not as good as lane11 and 12?
Thanks!
That sounds right Shuaichen, since the template is the only changing parameter. Interesting finding,
Abhishek
template quality is a difficult concept. In the early days of pcr we used to deliberately break up long genomic dna because smaller fragments melted easier so the early cycles of pcr worked better so sometimes degraded dna works better and can be a good thing as it also makes it more likely that the primer can find its annealing position ( ie not hidden in a large knot of dna. What may be happening is that your dna has inhibitors so using less dna means less inhibitor so better amplification. Anything that takes magnesium out of the reaction mix can be an inhibitor so EDTA, many plant products that mimic dna and co precipitate with it, smal degraded dna and even ntps in excess can also remove magnesium. If you get better amplification with less dna then inhibitors are a good bet and further purification may be called for although you have very good amplification with less template so just keep using less template.
Hi,
as you suggested it does indeed look like DNA quality/purity has an effect on your PCR yield. I guess the question is what you want to do in the next step. If you intend to sequence the amplicon, I would think that even the DNA yield shown in less intense bands is sufficient, so you might not need to worry too much about it. One thing you could try is to excise and extract the DNA of the less intense bands and re-amplify them. There is a Qiagen kit, called QIAquick. I have use that before and it worked pretty well for me.
Cheers, Oliver
200 ng is an unnecessarily large amount of template; it corresponds to 5.107 copies of the E. coli genome. Try 20 ng or 2 ng. This will have the advantage of diluting away any inhibitors that might be in the sample.
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