Hi Aidanmc

According to a place called “IDT” (Integrated DNA Technologies), nuclease-free TE pH 8.0 is best to store your amplicon

in (since DNA is more ‘stable’ at pH 8.0)

For your 'point 4':

It will be your calculation of the Molar extinction coefficient of your PCR product that is key ...

You will either need to calculate the molar extinction coefficient of your GAPDH product based on its sequence, using the dNTP pH 8 values (if measuring in TE pH 8) for each base (ABI values shown below) and add up all values to come up with the final sum value for an estimate, or, by using the “nearest neighbor” method which takes base order/stacking into account...

ABI values for deoxyribonucleotides (dNTPs) at pH 8.0:

dA = 15200 M-1cm-1

dC = 7050 M-1cm-1

dG = 12010 M-1cm-1

dT = 8400 M-1cm-1

So, if your sequence is ATTGCT... you would add 15200 + 8400 + 8400 + 12010 + 7050 + 8400 ... and so on for the sense strand, then do the same for the anti-sense strand until you get the final summed value for both strands - which would represent your molar extinction coefficient for your dsDNA GA3PDH PCR amplicon product sequence.

See more details on this calculation on p. 11 of the following:

http://cdn.idtdna.com/support/technical ... torage.pdfAside:

[And, remember, to the qPCR, a double-stranded product = 2 copies of target. So, whatever you are assessing (e.g. gDNA vs. transcriptome), be sure you remember this. gDNA is dsDNA, and transcriptome is ssRNA (thus single-stranded cDNA starting as the first strand)... in general. So, 1 copy of amplicon on its standard curve = 1 copy of gDNA on its standard curve since both go into the qPCR as dsDNA species. But, if using a dsDNA plasmid to help calculate copies of a transcriptomic target mRNA/cDNA, then one must remember that 1 copy of plasmid is actually 2 copies of target when comparing it to a transcriptomic target …]Anyway:

Once you have computed the extinction coefficient for your GA3PDH amplicon sequence, (let’s say you calculate it to be 2532360 L/mol*cm. And you diluted it in TE pH 8.0, and, let’s say, at a 1:10 dilution of your amplicon (after the NanoDrop has been blanked using TE pH 8.0) you get an A260nm absorbance reading of 6.3309 on a NanoDrop. Using the Beer-Lambert equation, A = ebc, we arrange it to the expression:

(10^6)*A

260nm*dilution/eb = c (in uM): so we calculate your undiluted amplicon solution concentration (in uM) to be: (10^6) * 6.3309 *10 / (2532360(M^-1cm^-1) * 1 cm) =

25 uMSo, if we find by NanoDrop that you have a 25 uM solution of your purified, dsPCR product:

since we know that uM = picomoles/ul ... and we also know that Avagadro’s number = 6.02214179 x 10^23,

then, since

25 uM = 25 picomoles/ul, and 25 picomoles/ul = 2.5 X 10^-11 moles/ul,

we can calculate that 2.5 X 10^-11 moles x 6.02214179 x 10^23 = # of dsGA3PDH amplicons/ul =

1.50553545 x 10^13 dsGA3PDH amplicons/ul (again "ds" here connotes "double-stranded")

This means that your 25 uM solution of purified dsGA3PDH amplicon in TE pH 8.0 contains 1.50553545 x 10^13 double-stranded GA3PDH amplicons in each ul. You can do your serial dilution calculations for your standard curve from here. And, the amplicon should work nicely as the material for a qPCR standard curve substance (as opposed to super-coiled plasmid DNA).

See if these calculations help you at all ... let me know if I lost you along the way here or not.

As you will find, it all comes down to how well you calculate the extinction coefficient of the amplicon is question

(e.g. which equation or equations you decide to believe, and use for that, is crucial ... everyone may do that

slightly differently -- perhaps Maniatis and Fritsch have a solid, unchanging idea on this ... e.g. Maniatais,

T., Fritsch, E. F., and Sambrook, J. (1982) Molecular. Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY ...) -- an exact approach (used by everyone everywhere) is a hard to dig up...

but, it would seem that the nearest neighbor method is probably the way to go. A good calculation of the molar

extinction coefficient calculation is key.

And always remember, the inverse of the molar extinction coefficient tells you what Molarity your solution

would be if your A260nm reading is exactly 1.0I find my way of doing these series of calculations fairly logical ... it is disconcerting that this seemingly basic maneuver

is so hard to find published in explicit/succinct terms isn't it?

JMG