Quantify DNA after PCR

[SBguy]

Hi there,
One problem in our lab is knowing whether we have had successful PCR amplification or not. We run a lot of PCR and our normal test is to run a gel afterwards to check that there's a band. This is time consuming especially when running lots of samples.

I was wondering if you know of any tips so DNA quantity can be measured *after* the PCR reaction to determine whether amplification occurred. It wouldn't have to be extremely accurate, but just give us the ability to tell the difference between "no amplification" and "amplification" without running a gel.

Ideas:
1. Nanodrop (don't have one, chime in if you have tried this)
2. EtBr UV spot test (haven't tried this, but have seen a few references to it)?
3. EtBr into tube, then put the tube in the imager and see if it glows, try to quantify this?
4. Other ideas?

Thanks!

SB Guy

[ashleyj722]

There is a method that uses Capillary Electrophoresis which can be used to quantify the amount of dsDNA formed but probably takes just as long as the Agarose gel. Could change to RT-PCR to quantify the DNA.

[LCMaul]

Yup, RT-PCR is the best way - I wish I could help you more on the gel.

[butters]

I did try it before. (don't ask)

1. Nanodrop
Well there is a significant increase for the positive and some increase for negative too. Since you see the word increase it mean you have to do a pre and post. Not a good idea if you have tonne of PCR product.

Do you expect more positive than negative? If no there are short cuts... ahem... tell me if you need the protocol. lolz. I shouldn't be teaching too much ahem... protocol. You still need to run gel, just a lot less.

2. This method is for estimation of DNA template. I think you will get headache trying to match it with your PCR product. Imagine you have to have a pre and post PCR. DNA template loaded may differ lots more dirty than anything else.

3. EtBr in tube in UV imager.
you will see the orange color every time. Why? you have DNA template.

4. Current none so far.

Importantly are your lab is governed by ISO? If yes it will be tricky to bypass... Not impossible.

[SBguy]

Hi all,
Thanks for the feedback!
I had no idea that the spot test was for estimating template. Have you used it for this?

Now, in regards to the EtBr in tube in UV imager, here's a paper that says it works (from 1992)! Have you tried it? Will you?
gene-quantification.de/higuchi-1992.pdf
(the site won't let me post links until 5 posts...just add the www back to the url)

Thanks!

SB Guy

[SBguy]

Hi all,
Any feedback on this? Thanks!!

[mmorgan]

Originally Posted by SBguy

Hi there,
One problem in our lab is knowing whether we have had successful PCR amplification or not. We run a lot of PCR and our normal test is to run a gel afterwards to check that there's a band. This is time consuming especially when running lots of samples.

I was wondering if you know of any tips so DNA quantity can be measured *after* the PCR reaction to determine whether amplification occurred. It wouldn't have to be extremely accurate, but just give us the ability to tell the difference between "no amplification" and "amplification" without running a gel.

Ideas:
1. Nanodrop (don't have one, chime in if you have tried this)
2. EtBr UV spot test (haven't tried this, but have seen a few references to it)?
3. EtBr into tube, then put the tube in the imager and see if it glows, try to quantify this?
4. Other ideas?

Thanks!

SB Guy

I think if you're looking for a quick, dirty and cheap assay EtBr into the tube or onto spots onto a surface you can put on a UV box is an OK idea.

Of course the primers will give flourescence but if you have a control reaction without template I expect you would see a difference since dsDNA is much brighter than ssDNA when stained with EtBr. The template fluorescence should be negligible relative to the primers and the amplicon.

The Nano-Drop is a great piece of equipment but I don't know how useful it would be for direct assay of a PCR reaction. From my own experience the Nano-Drop reading gets skewed by free dNTPs so you might have to go through a clean up column. Of course if you're doing so many PCRs that running gels is too time consuming I'm sure doing a clean up column for each reaction is out of the question.

The only problem with not running a gel is that you have no idea if you have amplified your intended target or if you've amplified a non-specific band.

[davidflora]

[Only registered users see links. ]The quantification strategy is the principal marker in gene quantification. Generally two strategies can be performed in real-time RT-PCR. The levels of expressed genes may be measured by absolute or relative quantitative real-time RT-PCR. Absolute quantification relates the PCR signal to input copy number using a calibration curve, while relative quantification measures the relative change in mRNA expression levels.