Upload
sachin-subba
View
807
Download
3
Embed Size (px)
Citation preview
DNA FINGERPRINTING
MATERIALS & METHODS
.
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
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
DAY 10.15g - 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
DAY 2Centrifuge at 12000rpm for 12mins at
4 C
Collect pellet & allow to air dry for 10mins
After air dry add 800of TE buffer
Add 6 of Rnase & incubate at 37 C for 30mins in the bath
After incubation add equal vol of phenol: chloroform: isoamly alcohol (25:24:1)
Centrifuge at 12000rpm for 12mins at R.T
After centrifuge we get three layer
Collect the upper layer & transfer to 2ml vials
Add equal vol of chloroform : isoamyl alcohol & mix it gently
Centrifuge at 12000rpm for 12mins at R.T
Collect upper layer & transfer to 2ml vials
Add 0.1v 3m sodium acetate & mix it well
Add full vol of absolute ethanol & mix it well
Store at -20°C overnight
DAY 3
Mix & centrifuge at 12000rpm for 12mins at 4°C
Collect the pellet & then add 1.5ml 70% ethanol
Dissolve pellet gently
Centrifuge at 12000rpm for 12mins at 4°C
Collect pellet & air dry for 10 – 15 mins
Add TE buffer
Dissolve the pellet gently
Prepare 0.8% of agarose gel
Load the 10μl genomic DNA
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
Seal the edges of clean casting tray
Prep sufficient buffer (1x TAE ) to fill electrophoresis tank to cast the gel
Prepare the soln of agarose in electrophoresis buffer at 0.8% conc
When molten has cooled add ETBR
Choose appropriate comb for forming the slots in gel & pour soln
Allow the gel to set completely, remove the comb & tape carefully
Mount the gel in the electrophoresis tank
Then pour the electrophoresis buffer to cover the gel to a depth of 1mm.
Slowly load the sample mixture into the slots using disposable micropipette at 50-100v
sample or dye is migrated a sufficient distance through the gel, turn off the electric current
Examine the gel by UV light and photograph the gel
SPECTROPHOTOMETRIC QUANTIFICATION OF DNA
REQUIREMENTS :
UV spectrophotometer
TE buffer
DNA sample
Micropipette
Absolute Ethanol
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
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
S
.
N
o
DNA sample
volume(µl)
Positive
control (µl)
dNTPs (µl) Buffer (10x)
(µl)
Forward
primer (µl)
Reverse
primer (µl)
Taq
polymerase
(µl)
Sterile
water (µl)
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
Reaction components
QUALITY PCR PROGRAMHeated 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
AMPLIFICATION OF DNA USING RAPDREQUIREMENTS :
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
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
PROCEDURE:
Set up the following reaction mixture (25 l) in the same order.
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
UREA POLYACRYLAMIDE GEL
ELECTROPHORESISREQUIREMENTS :
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
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.
Electrophoresis of the DNAPre-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.
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.
PROCEDURE :
Place the gel in 5 volumes of a mixture of 30% ethanol and 10% acetic acid.
Incubate the gel for 3 hours or overnight with shaking at room temperatures.
Remove the ethanol / acetic acid solution and add 5 gel volume of 30% ethanol.
Incubate for 30minutes at room temperatures with shaking. Repeat this step twice.
Remove the ethanol solution and add 10 gel volume of deinonised water.
Incubate for 10minutes at room temperatures with shaking. Repeat this step twice.
Remove the deionised water and add 5 gel volume of 0.1%silver nitrate solution.
Incubate for 30minutes at room temperatures with shaking.
Remove the silver nitrate solution and wash the gel for 20seconds under a stream of deionised water.
Add 5 gel volume of a mixture of 2.5%sodium carbonate and 0.02% formaldehyde.
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.
THANK YOU
-SACHIN SUBBA
DEPT OF BIOTECH
CMRIT, BANGALORE