I'm upload the slide into ''estimate the DNA by Diphenylamine method''.

1. ESTIMATION OF DNA BY DIPHENYLAMINE
METHOD
Presented by,
K.Jeevithakumari.
MSc biotechnology
Presented to,
Dr.Immanuel joshua jeba singh

2.  DNA is a Deoxyribonucleic acid that carries the genetic
information in the cell and is capable of self-replication and
synthesis of RNA.
 DNA consists of two long chains of nucleotides twisted into a
double helix and joined by hydrogen bonds between the
complementary bases adenine and thymine or cytosine and
guanine.
 It acts as the genetic material &Carries the genetic information
 The sequence of nucleotides determines individual hereditary
characteristics.

3. To determine the concentration of a given
DNA sample using diphenylamine method.
When DNA is treated with diphenylamine under the acidic
condition a bluish green colored complex is formed.This
reaction is given by 2 deoxypentose in general. In acidic
solution deoxypentose are converted into a highly reactive β
hydroxyl leavulinic aldehyde which reacts with diphenylamine
gives bluish green colored complex. The colour intensity was
measured using a red filter at 595nm.

4. A UV/Visible spectrophotometer,Vortex mixer, Weighing
balance,Water bath
Diphenylamine reagent, Calf thymus DNA,Glacial acetic
acid, Concentrated sulfuric acid
Pipettes, Pipette tips,5 ml glass pipette, 100 ml measuring
jar,250 ml amber colored glass bottle, Test tubes, Caps for
glass tubes, Distilled water, Quartz or glass cuvettes

5. Weigh 1g of diphenylamine and transfer it into a 250 ml amber
coloured glass bottle. Add 100 ml glacial acetic acid and shake
well to achieve complete dissolution. Add 2.5 ml of
concentrated sulfuric acid. Store the reagent in dark at 2 –
8°C.
Dissolve 1g of diphenylamine in 100 ml of glacial acetic acid
and 2.5 ml of concentrated H2SO4. This solution must be
prepared fresh
0.5 mol/litre NaCl; 0.015 mol/litre sodium citrate, pH 7.

6. . Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working
standard in to the series of labeled test tubes.
. Pipette out 1 ml of the given sample in another test
tube.

7. . Make up the volume H2O to 1 ml in all the test tubes. A tube with 1
ml of distilled water serves as the blank.
. Now add 2 ml of DPA reagent to all the test tubes including the test
tubes labeled 'blank' and 'unknown'.
. Mix the contents of the tubes by vortexing / shaking the tubes and
incubate on a boiling water bath for 10min.
. Then cool the contents and record the absorbance at 595 nm against
blank.
. Then plot the standard curve by taking concentration of DNA along
X-axis and absorbance at 595 nm along Y-axis.
. Then from this standard curve calculate the concentration of DNA
in the given sample.

8. No of
tubes
Volume of
std(mg/ml)
Volume of
H2o
Volume of
DPA
reagent
Con of std OD at
595nm
S1 0.2 0.8 2 2
S2 0.4 0.6 2 4
S3 0.6 0.4 2 6
S4 0.8 0.2 2 8
S5 1 - 2 10
B - 1 2 -
T 1 1 2 -

9. Volume of std(mug/ml
ODat595nm(mug/ml)
Y
x

10. Concentration of
unknown sample
The given unknown sample contains ----µg/ml.
= Test OD value/standard OD value*concentration of std