Hi,

If the concentration is measured on NanoDrop then it's possible because it measures DNA conc. not so precisely.

DNA may not have been dissolved properly in stock solution and is of poor quality.

Dear Dr. Ali

Thanks for your quick reply

What should I do to improve my PCR reaction. Are these DNA useless?

Dear Amit,

Have you tried amplifying 100ng/ul DNA dissolved in TE buffer?

I dont think adding water or TE is the problem.

You cant say these samples useless just you have to do little bit standardization of your protocol.

Dear Pankaj

Thanks for your reply

I have already increased the DNA conc. in PCR. Yes I have tried amplifying 50ng/ul DNA dissolved in TE buffer. But it didnot get amplify, but the DNA of 100ng/ul dissolved in water (it was made to 100ng/ul with water out of 500ng/ul stock dissolved in TE buffer). Since the conc. of DNA (50ng/ul) dissolved in TE ,was less so I directly used it in the PCR and it didnot amplify.

Amit, Try with Real Time PCR. Where you can get amplification at lower DNA concentration also.

Sorry pankaj, we do not have real time PCR. Is there any other methods, which can help?

You might have reached the lower limit of detection for this primer pair and genomic DNA. With cloned sequences you can get orders of magnitude lower. You have a lot of other sequences in there which cannot be amplified, but is of course part of the DNA concentration measurement. You can repeat your measurement, it is not necessarily unprecise, the lower detection limit of the Nanodrops is 2ng/ul.

TE buffer doesn't interfere with PCR in my experience since you typically add only very litte amounts of DNA (volumewise) to the PCR reaction.

I don't see the problem of using more DNA in your PCR unless you have very little material.

@Pankaj: The only difference between standard PCR and real-time PCR is the presence of a fluorophor which binds to dsDNA, which enable the detection during the reaction. You can only see reaction products earlier, since this is muc more sensitive than a standard agaroe gel. Reactionwise there is no difference - so if there is no amplification in standard PCR, there is most likely no amplification in realtime PCR, too.

it is a concentration problem but it might also be interference with EDTA. PCR in some cases requires Magnesium ions and EDTA chelates those ions and does not allow them to be available. I have found out that water works like a charm for diluting concentrated DNA samples.

I always use TE buffer to dissolve plasmids and DNA (because the stability for storage is better) and had never any problems. This is actually no wonder, since the EDTA concentration is very small in your PCR reaction. Let the reaction volume be 20ul and you add 1ul of DNA solution in TE buffer (which contains EDTA in a concentration of 1mM), then you will chelate 50nM (1mM/20ul) magnesium away. Since a typical PCR reactions contains between 2 and 3mM magnesium ions, this effect is not noticable. In high concentrations this picture looks different, since magnesium ions are absoutely vital for the function of the reaction. BTW: Very high primer concentrations have the same effect, since the DNA forms a complex with magnesium.

But if it always works with 100ng and doesn'T with 50, I wouldn't really waste time with it and use 100ng for PCR. We have genomic DNA here so most of the DNA (99% or so) present in the solution cannot be amplified, since its the wrong sequence.

TE buffer has EDTA which will chelate the magnesium ions. Mg2+ ions are required by the polymerase during extension. Since you have diluted the DNA in TE buffer, the EDTA is causing problem with the extension.

@Bhairavi: Its not problem - thats my practical experience and that of many other people. Its not, because the EDTA concentration in you PCR is ways to small to have a real inhibitory effect.

The concentration of the EDTA in the TE-Buffer is 1mM and 1mM of EDTA can chelate 1mM of ions. One of my typical PCR reactions has 25ul, containing 1ul of DNA template. This means the concentration of the EDTA in the reaction is 40nM, meaning it can chelate 40nM of magnesium. This would lower the concentration of 2,5mM magnesium which we often use down to 2,46mM. The impact of a improper calibrated pipette (with an error of lets say 10%) is ways bigger. So don't worry about a little EDTA, the benefit of stabilizing the DNA with a higher pH is much bigger.