Variants

Dosage of very low DNA concentrations Using OliGreen®



Kit Quant-It™OliGreen® from Molecular Probes.
You need Abgene(#AB-0700) ThermoFast 96-well plates + Ultra Clear Cap Strips (#AB-0866)
Goal is to dose small amounts of DNA such as after Chromatin IP.

Dilute TE 20X from kit H20 millipore to obtain TE 1X.
Prepare SO (standard oligo mix) = 1 μl of oligo 100 μg/ml from kit + 99 μl of TE 1X
Prepare R mix = 3 μl of oliG from kit + 600 μl of TE 1X (PROTECT FROM LIGHT)

In Thermo-Fast plates, prepare the following standards :

500 ng/ml :    100 μl SO
250 ng/ml:    50 μl TE + 50 μl SO
50 ng/ml:    90 μl TE + 10 μl SO
5 ng/ml:    99 μl TE + 1 μl SO
0 ng/ml:    100 μl TE
Your sample:    99 μl TE + 1 μl sample

Go to the Stratagene qPCR machine : MxPro -> Quantitative Plate Read -> to the question box « Wish SYBR Green ? » answer « no » -> Import -> (get a file from LN folder) -> Adapt if necessary (well type : standard or unknown, FAM and only FAM should be checked) -> Set Read Properties : 25°C, 3 reads.

Once you’re ready to run it, add 100 μl of R to every tube. Wait 2 minutes while protecting the tubes from light.

Load in the machine and run.

Dernière mise à jour : ( 10-12-2007 )