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Estimation of DNA fragmentation by diphenylamine (DPA) assay
37
LAB 4:
Estimation of fragmented DNA
by diphenylamine
by.. Alaa Alahmadi
Procedure..
200 µl of supernatant + 200 µl of 10% TCA
Place tubes in 90°C water bath for for 20 min
How to read agarose gel electrophoresis results ??
Loading Samples and Running on Agarose Gel
Add 2 µLof loading dye to 5 µL of DNA
* Once solidified, place the agarose gel into the gel tank. Fill gel tank with 1xTBE until the gel is covered.
Turn OFF power, then carefully carry the gel (in its holder if possible) to the UV light-box to visualize your DNA fragments ‘bands’.
Close the gel tank, switch on the power source and run the gel at 80-150V until the dye line is approximately 75-80% of the way down the gel.
*Carefully load a molecular weight ladder (marker) into the first lane of the gel.
* Carefully load your samples into the additional wells of the gel.
Centrifuge for 10 min, then discard pellet
200 µl of supernatant + 800 µl of DPA
Calculation...
Place tubes in 100°C water bath for 10 min.
OD of supernatant
X
100
% of DNA fragmentation =
Cool the tubes and measure the absorbance
at 595 nm.
OD of supernatant
+
OD of pellet
* Blank is a mixture of 200 µl lysis buffer+ 200 µl of TCA +800 µl of DPA
THANK YOU !!
What is DPA ?
* Diphenylamine is an organic compound
* A diphenylamine indicator is often used for the detection of DNA.
* DPA assay is quantitative test that used in biochemistry.
Principle
1) Boiling of DNA under acidic condition,