DAY 10.15g - 0.3g in 700- 900μl of CTAB buffer200 each time & crush with the help of Motor & pestelTransfer to 2ml of vials & incubate T 50°C for 15mins in water bathAfter incubation add equal vol of chloroform: isoamyl alcohol (24:1)Incubate at 37°C for 30mins in shakerCentrifuge at 12000rpm for 12mins at R.TCollect the upper layer & transfer t 2ml vials.Add 0.5v 5m Nacl & mix it well & then add full vol of isopropanolStorage for -20°C overnight
DAY 2 After centrifuge we get three layerCentrifuge at 12000rpm for 12mins at 4 C Collect the upper layer & transfer to 2ml vialsCollect pellet & allow to air dry for Add equal vol of chloroform : isoamyl alcohol 10mins & mix it gentlyAfter air dry add 800of TE buffer Centrifuge at 12000rpm for 12mins at R.T Collect upper layer & transfer to 2ml vialsAdd 6 of Rnase & incubate at 37 C for 30mins in the bath Add 0.1v 3m sodium acetate & mix it wellAfter 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 overnightCentrifuge at 12000rpm for 12mins at R.T
DAY 3Mix & centrifuge at Collect pellet & air dry for 12000rpm for 12mins 10 – 15 mins at 4°C Add TE bufferCollect the pellet & then add 1.5ml 70% ethanol Dissolve the pellet gentlyDissolve pellet gently Prepare 0.8% of agarose gelCentrifuge at 12000rpm for 12mins at 4°C Load the 10μl genomic DNA
AGAROSE GEL ELECTROPHORESISREQUIREMENTS : 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 Mount the gel in the electrophoresis tankPrep 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 50-100vWhen molten has cooled add ETBR sample or dye is migrated a sufficient distance through theChoose 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 gelAllow the gel to set completely, remove the comb & tape carefully
SPECTROPHOTOMETRIC QUANTIFICATIONOF DNAREQUIREMENTS : 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 PCRREQUIREMENTS : 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
QUALITY PCR PROGRAMHeated lid 110ºC Pre- heated lid off Pause- off Initial denaturation- offLoop 1 (initial denaturation) No. of cycles 1 Segment 94ºC 3minutesLoop 2 No. of cycles 30 Segment 94ºC 30sec Segment - 55ºC - 30sec Segment - 72ºC - 1minuteFinal extention 72ºC - 5minutesFinal 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
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
All those mentioned ingredients are mixed and prepared for the total no of reactns including a blank with a particular primer excluding templateThe 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 tubeThe contents of the tube are mixed with a brief spin and transferred to PTC 200 thermal cyclerThe program with following conditions is selected for the amplificationNumber cycles 30Segment 94.0ºC 1minuteSegment 35.0ºC 1minuteSegment 72.0ºC 1minute
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.
Incubate for 10minutes atPROCEDURE : room temperatures with shaking. Repeat this stepPlace 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 ofIncubate the gel for 3 hours or 0.1%silver nitrate solution. overnight with shaking at room temperatures. Incubate for 30minutes atRemove the ethanol / acetic acid room temperatures with solution and add 5 gel volume shaking. of 30% ethanol. Remove the silver nitrateIncubate 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.
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 eachThe 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