Hello,
can any body please tell me how to improve the concentration of PCR product?or how to increase the concentrations of gel purified/gel extracted product?
my preference is to improve the concentration of PCR product? if any body give me some good suggestion about this u will be very very thankful.........
Hi Fahim,
without anymore specifics some of my suggestions might not be the best but here they are:
Optimize your PCR reaction. You could try modifications on your reaction such as modifying the Mg concentration, including some aditives such as DMSO, or spermine if your template is GC rich. You could also modify the annealing temperature, again if you haven't optimize it yet. Make sure that you don't add too much template and that you have enough primers and dNTPs.
Increase the cycle number. This is not the best approach as it can increase the possibilities of changes in your product but if you are using a good enzyme it could work.
Nested PCR. If your PCR is specific you could do a nested PCR, basically this is a PCR done using the PCR product as template.
Run extra PCRs. You could run 5-6 PCRs and then load them together in a gel with a big comb (you can use tape to combine wells using regular comb). Then you can purify it from the gel. I would recomend you to use a low concentration gel as the purification would be better. Alternatively you can precipitate the PCRs with ethanol and then run it in a gel to remove template, etc.
Clone it. Depending on what the purpose is, you could just clone it and then just amplify it. A TOPO vector would work like a charm for this.
Hope this helps,
Ezequiel
I want to assume that your aim is to get a reasonable quantity of the gel purified product and you thought the best way to do so is to increase the concentration of your PCR product.
1. The concentration of the gel purified product largely depends on the band intensity. If your band is not very thick, you may need to optimise your PCR reaction to achieve optimal amplification by following the suggestions made by Ezequel.
2. If your band is intensed but you keep having low concentration of the gel purified product, then
a. Make sure you excise your gel as close to the band as possible to avoid excess gel mass.
b. Depending on the kit you are using, ensure that you solubulise your gel adequately by heating in a heatblock for 10 min (or more if the gel is more than 1%).
c. Pre-heat the elution buffer at 55-60 degrees for at least 30 min and use immediately to elute your DNA from the spin column. Dont forget to allow the column to stand for atleast 3 minutes after the addition of the elution buffer before the final centrifugation.
Hope it helps
Bashir
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