DNA quantification - How to quantify my PCR product before sequencing?? (Sep/08/2004 )

Hi,

Anyone can tell me how can i quantify my PCR product directly from the agarose gel ( quantification before sequencing)?, i think that it is possible by compare the intensity of my PCR product with the intensity of a DNA marker ! but what is the formula ??

thanks for your help

selim

Hi Selim,

Usuall I won't bother measuring the concentration of my PCR products before sent for seqeuncing, I just estimate the conc. by the intensity of my bands. If you do want to know the conc., you can do a spot test which is very easy. Here you can find a good protocol for this test http://www.unizh.ch/botinst/Cyto_Website/s...n/spotTest.html

Hi,
do you purify your samples from the gel before sequencing? Why not spec the DNA in that case, you can use a small aliquot of diluted DNA so you don't waste your sample.
As far as markers are concerned: as long as you know what amount of marker you load and you have all the band sizes of the marker from your manufacturer, you can work out the amount of DNA in ng of each band, and by comparing the intensities work out the APPROXIMATE amount of your PCR product. For example, total base pair sum of marker SPP1 is 43787 bases (all bands added together). Then teh top band, which is 8557 bases represents 8557/43787= 0.195 of DNA. If you load 500ng of marker, then your top band will have 0.195X500= 97ng of DNA in it.
Ok, hope this helps

When you analyze youre PCR product on a agarose gel,
you can have a MassRuler, from fermentas i think, as a marker.

With this marker you can see it youre PCR product has the correct fragment size, and by compairing the intensity of the band in the marker to the PCR band you can with a known concentration ruler estimate the concentration of the sample.

/Nina

Hi,
Along the same lines as Selim's question... if I need to know the exact concentration of my PCR product following amplification (for use as target fragments on an oligo microarray) what is the best procedure for accuracy?

I know that I can do gel extraction, but this is not very accurate is it? How accurate is spectrophotometry?

In this study, it is essential that I know exact concentrations!

Thanks much,
Noel

Re Noel's question,
you are right, gels are only approximate. Spectrophotometry is usually good, as long as your technique is also good, ie good pipetting if you are using a dilution (I normally use 1/50) to measure concentration, clean tubes for blanking, etc. However the spec will give you total nucleotide concentration, which might be prone to error in some cases, eg. if your product is degraded, etc.
what's the experiment you need exact conc for?

Hi

You can also scan your gel, and then using the software that goes with the scanner count the pixels in your band and in one of the marker's. Knowing the DNA quantity in the marker'band you chose, you can estimate the quantity of DNA in your band.
Now I don't know how accurate this method is, but I use it to adjust the RNA concentration before doing semi-quantitative RT-PCR, so I think this should work