Some colleagues tell to me to dry the sample to something like 20 microlitres so as to increase chances of picking out a bacterial DNA during PCR since I am using clinical samples to extract bacterial DNA for PCR.
I am not to that since drying samples will lead to a highly concentrated DNA sample that would hinder PCR since too much DNA template hinders PCR.
No, don't do that. If you dry your sample you will significantly increase the concentration of the salts composing your elution buffer (probably Tris and possibly EDTA). This will possibly result in inhibition of your PCR. Instead, try first the PCR without any drying as in most cases PCR can be a very efficient reaction and can work even with miniscule amounts of DNA template.
No, don't do that. If you dry your sample you will significantly increase the concentration of the salts composing your elution buffer (probably Tris and possibly EDTA). This will possibly result in inhibition of your PCR. Instead, try first the PCR without any drying as in most cases PCR can be a very efficient reaction and can work even with miniscule amounts of DNA template.
I agree with Yehu and I suggest you to elute in a smaller amount of diluited elution buffer (1:10 should be enough).
I don't think you need to dilute, you should quantificate your sample and that way you can know the exact concentration you are using in order to use more, or even dilute it to a work solution that you use especifically on your PCRs... At least that's what I do
I agree with Sarah, you don´t need to dilute, quantify your sample and run your PCR. based on your results you must adjust concentration and parameters.
I agree with the all, but instead 200 ul,you can use 80-100 ul eluted buffer. This is will be better to obtain good PCR results, but do not try to dry your samples.
Elution volume does not matter very much. It depends on how much DNA you get. But if your 200 ul only contain 1-2 ng or so and PCR does not work-elute in water and then reduce the volume by drying.
Hi , i didn't understand why you used 200µl of elution buffer, you can use only 100µl, even if you did not i think it doesen't matter just you have to know exactely the concentration of you samples, by which you could know the sufficient quantity that you should use for your PCR.
Sarah so you mean once i run the PCR and its unsuccessful
, then i can come back and adjust the initial concentration I had...
Younes I did that since at first, I wanted to use the Qiagen protocol with no modifications, it is a starting point, so that later i may know where to alter. What modifications do you employ for your protocol?
An alternative to alcohol precipitate your DNA if you already have it in 200ul volumes and reconstitute in a lesser volume. However, as the above have said, you need to know the concentrations at which you get amplification. You could use a human DNA sample spiked with diff concentrations of bacterial DNA to see at which concentrations you get an amplification in your sample
You can also try eluting with smaller volume, say 50 - 100 microlitres if you don't get a good result with the 200.
You first must be measure the DNA concentration and after and then adjust the concentration for your pcr. Usually the concentration
what should I do after I check concentration and I realise concentration is low but want to recover the DNA into a smaller volume with the aim of increasing the concentration.
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