The document describes the process of DNA fingerprinting of 6 varieties of chili plants. It involves collecting samples from different varieties of chili plants and extracting DNA from the samples using the CTAB method. The extracted DNA is quantified using a spectrophotometer and amplified using PCR and RAPD. The amplified DNA is separated and visualized on an agarose gel and urea polyacrylamide gel through electrophoresis. The gels are then stained using silver staining for visualization and documentation of DNA bands.

1. DNA FINGERPRINTING
MATERIALS &
METHODS
.

2. SAMPLE COLLECTION
6 verities of chili plants were collected from
G.KV.K agri university, Bangalore. The
samples are stored at 4⁰c.
 Guntur
 Sarca aroka
 Samruthi
 Indm kaddi
 Capsium-L
 SBL-C

3. DNA EXTRATION
(CTAB Method)
REQUIREMENTS:
 CTAB extraction buffer
 0.3M Sodium Acetate.
 Phenol : Chloroform : Isoamyl Alcohol
 Isopropanol
 Ribonuclease
 70% Ethanol
 5M Sodium Chloride
 TE buffer
 Chloroform : Isoamyl Alcohol

4. DAY 1
0.15g - 0.3g in 700- 900μl of CTAB buffer
200 each time & crush with the help of Motor & pestel
Transfer to 2ml of vials & incubate T 50°C for 15mins in water bath
After incubation add equal vol of chloroform: isoamyl alcohol (24:1)
Incubate at 37°C for 30mins in shaker
Centrifuge at 12000rpm for 12mins at R.T
Collect the upper layer & transfer t 2ml vials.
Add 0.5v 5m Nacl & mix it well & then add full vol of isopropanol
Storage for -20°C overnight

5. DAY 2
After centrifuge we get three layer
Centrifuge at 12000rpm for 12mins at
4 C
Collect the upper layer & transfer to 2ml vials
Collect pellet & allow to air dry for Add equal vol of chloroform : isoamyl alcohol
10mins & mix it gently
After air dry add 800of TE buffer Centrifuge at 12000rpm for 12mins at R.T
Collect upper layer & transfer to 2ml vials
Add 6 of Rnase & incubate at 37 C for
30mins in the bath
Add 0.1v 3m sodium acetate & mix it well
After incubation add equal vol of Add full vol of absolute ethanol & mix it well
phenol: chloroform: isoamly
alcohol (25:24:1)
Store at -20°C overnight
Centrifuge at 12000rpm for 12mins at
R.T

6. DAY 3
Mix & centrifuge at Collect pellet & air dry for
12000rpm for 12mins 10 – 15 mins
at 4°C
Add TE buffer
Collect the pellet & then
add 1.5ml 70% ethanol
Dissolve the pellet gently
Dissolve pellet gently Prepare 0.8% of agarose
gel
Centrifuge at 12000rpm
for 12mins at 4°C
Load the 10μl genomic
DNA

7. AGAROSE GEL ELECTROPHORESIS
REQUIREMENTS :
 Electrophoretic unit
 Methanol
 Electrophoretic medium
 Solid support (1.2%agarose)
 Gel loading buffer
 Gel-sealing tape
 Micropipette with disposable tips
 Microfuge vials
 Sample
 Uv Transilluminator
 Stain

8. Seal the edges of clean casting tray Mount the gel in the electrophoresis
tank
Prep sufficient buffer (1x TAE ) to fill
electrophoresis tank to cast the Then pour the electrophoresis
gel buffer to cover the gel to a depth
of 1mm.
Prepare the soln of agarose in
electrophoresis buffer at 0.8% Slowly load the sample mixture into
conc the slots using disposable
micropipette at 50-100v
When molten has cooled add ETBR
sample or dye is migrated a
sufficient distance through the
Choose appropriate comb for gel, turn off the electric current
forming the slots in gel & pour
soln
Examine the gel by UV light and
photograph the gel
Allow the gel to set
completely, remove the comb &
tape carefully

9. SPECTROPHOTOMETRIC QUANTIFICATION
OF DNA
REQUIREMENTS :
 UV spectrophotometer
 TE buffer
 DNA sample
 Micropipette
 Absolute Ethanol

10. PROCEDURE:
Prepare a known dilution of DNA sample in the TE
buffer, which is used to dissolve the DNA sample.
Calibrate the spectrophotometer for blank using TE
buffer.
Record the OD of the sample at 260nm and 280nm.
Calculate the concentration of DNA in the sample
using the Relation

11. QUALITY PCR
REQUIREMENTS :
 Thermo stable Taq DNA polymerase
 dNTP mix (10mM)
 Chili genomic DNA
 Sterile distilled water
 PCR buffer (10x)
 Forward primers and reverse primer specific to
positive control
 Micropipettes of different ranges

12. Reaction components
S DNA sample Positive dNTPs (µl) Buffer (10x) Forward Reverse Taq Sterile
. volume(µl) control (µl) (µl) primer (µl) primer (µl) polymerase water (µl)
N (µl)
o
1 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
2 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
3 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
4 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
5 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
6 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

13. QUALITY PCR PROGRAM
Heated lid 110ºC
Pre- heated lid off
Pause- off
Initial denaturation- off
Loop 1 (initial denaturation)
No. of cycles 1
Segment 94ºC 3minutes
Loop 2
No. of cycles 30
Segment 94ºC 30sec
Segment - 55ºC - 30sec
Segment - 72ºC - 1minute
Final extention 72ºC - 5minutes
Final hold 10ºC

14. AMPLIFICATION OF DNA USING RAPD
REQUIREMENTS :
 Thermostable Taq DNA polymerase
 dNTP mix (10 mM)
 Template DNA
 Sterile distilled water
 PCR buffer (10x)
 Oligonucleotide primers
 Ice bucket
 Eppendorff vials
 Micropipettes of different ranges
 Thermal cycle

15. PROCEDURE:
Set up the following reaction mixture (25 l) in the same order.
Ingredients Volume to be taken
Template DNA 10.0μl
dNTPs 2.5μl
PCR buffer 2.5μl
Primers 1.0μl
Taq DNA polymerase 0.75μl
Sterile water 8.25μl
Total 25μl

16. All those mentioned ingredients are mixed and prepared for the
total no of reactns including a blank with a particular primer
excluding template
The calculated volume of masters mix ix then transferred to
labeled PCR tubes with template source and primer.
Finally 0.33µl of Taq DNA polymerase is added to each tube
The contents of the tube are mixed with a brief spin and
transferred to PTC 200 thermal cycler
The program with following conditions is selected for the
amplification
Number cycles 30
Segment 94.0ºC 1minute
Segment 35.0ºC 1minute
Segment 72.0ºC 1minute

17. UREA POLYACRYLAMIDE GEL
ELECTROPHORESIS
REQUIREMENTS :
 Vertical electrophoresis unit
 Urea 7M
 Acrylamide 40%
 10x TBE (Tris Borate EDTA) buffer
 10%Ammonium Per Sulphate (APS)
 Tetra Ethyl Methylene Diamine (TEMED)
 Gel loading dye
 Autoclaved distilled water

18. PROCEDURE :
Preparation of gel (50ml)
Weigh 9.08g of urea and dissolved by heating in about 15ml
autoclaved distilled water.
Add 6.25ml of 40% acrylamide and 5ml of 10x TBE buffer.
Make up the volume to 50ml with autoclaved distilled water.
Add 350μl of APS and 35l of TEMED and mix well.
Immediately transfer the gel into the previously arranged
vertical electrophoresis unit.

19. Electrophoresis of the DNA
Pre-run the gel for about one hour at 100V.
To the PCR sample add 4.2l of gel loading dye.(Xylene
Cyanol).
Boil the samples for 10minutes at 85-90C.
Immediately chill the sample in ice for 2minutes.
Spin the sample at 3000rpm for 2minutes and load in top
the gel.
The electrophoresis is carried out at 150V tll the dye front
reaches the bottom of the base plate, the plates are
cooled with an ice pack during the run to prevent over-
heating.

20. SILVER STAINING
REQUIREMENTS :
 Gel container
 Shaker incubator
 10% acetic acid
 de-ionised water
 autoclaved double distilled water
 silver nitrate solution
 2.5% sodium carbonate and 0.02% formaldehyde.

21. Incubate for 10minutes at
PROCEDURE : room temperatures with
shaking. Repeat this step
Place the gel in 5 volumes of a twice.
mixture of 30% ethanol and
10% acetic acid.
Remove the deionised water
and add 5 gel volume of
Incubate the gel for 3 hours or 0.1%silver nitrate solution.
overnight with shaking at
room temperatures.
Incubate for 30minutes at
Remove the ethanol / acetic acid room temperatures with
solution and add 5 gel volume shaking.
of 30% ethanol.
Remove the silver nitrate
Incubate for 30minutes at room solution and wash the gel
temperatures with shaking. for 20seconds under a
Repeat this step twice. stream of deionised water.
Remove the ethanol solution Add 5 gel volume of a mixture
and add 10 gel volume of
deinonised water. of 2.5%sodium carbonate
and 0.02% formaldehyde.

22. Incubate at room temperature with shaking. Bands will start
appearing slowly.
Incubate until band appears.
Stop the reaction by washing with 1% acetic acid.
Wash several times with deionised water for 10 minutes each
The gel might now be observed over an illuminating source of
white light for better result and documented.
For preserving the gel, place it in 20ml of a 20% glycerol solution.
Keep the gel between two layers of gelatin [aper and dry for 3
days at 37ºC.

23. THANK YOU
-SACHIN SUBBA
DEPT OF BIOTECH
CMRIT, BANGALORE