DNA Extraction:

DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in which it normally resides. Extraction of DNA is often an early step in many diagnostic processes used to detect bacteria and viruses in the environment as well as diagnosing disease and genetic disorders.

How does it work?

1. Break open (lyse) the cells or virus containing the DNA of interestThis is often done by sonicating or bead beating the sample. Vortexing with phenol (sometimes heated) is often effective for breaking down protienacious cellular walls or viral capsids. The addition of a detergent such as SDS is often necessary to remove lipid membranes. 2. DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases. 3. DNA is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added. 4. Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet. 5. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE. 6. Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light.

Instrumentation used in DNA Extraction:

Bead beater is used in the breaking apart or "lysing" of cells in the early steps of extraction in order to make the DNA accessible. A centrifuge such as this can spin at up to 15,000 rpm to facilitate separation of the different phases of the extraction. It is also used to precipitate the DNA after the salts are washed away with ethanol and or isopropanol. A gel box is used to separate DNA in an agarose gel with an electrical charge.

1. Extraction of Mycobacterial DNA: Materials

Instruments Safetygloves biosafety cabinet sterile tubes microcentrifuge vortex micropipettes

 Reagents

autoclave uv -illuminator

NaOH sol, sorensons buffer, 10X TE Buffer

Reagent Preparation: (a) 1M NaoH sol NaOH Nuclease free water 4g 100ml

4g of NaOH was dissolved in nuclease free water and the volume was made upto 100ml.Then autoclaved and stored at room temprature. (b)Sorensons buffer Solution A: 0.2M NaH2PO4.2H2O 35.61g

Solution A was prepared by dissolving 31.21g of NaH2PO4.2H2O in distilled H2O to make volume upto 1.0 L. Solution B: 0.2M NaH2PO4.2H2O 31.21g

Solution B was prepared by dissolving 31.21g of NaH2PO4.2H2O in distilled H2O to make volume upto 1.0L.

pH
6.8

solnA
24.5ml

solnB
25.5ml

Buffer was orepared by mixing two solutions and adjusted pH to 8.3 by adding HCl drop wise.Then autoclaved and stored at room temprature (c)10X TE Buffer(100mM)

Trisma base EDTA Water

1.21g 0.372g 100ml

1.21g of Trisma base and 0.372 of EDTA were dissolved in distilled water and adjusted the pH to 8.3 by adding HCl drop wise.Distilleded water was added to make the volume upto 100ml.Then autoclaved and stored at room temperature.

Methodology:
Equal volume of sputum and 4% NaOH were added in a vial,vortexed for 1 minute and incubated at 370C for 30 minutes in the water bath. Then supernatant was centrifuged and flicked off. 2ml Sorensons buffer was added to wash the pellet and repeated the washing for 3 minutes. 300 ul 50mM TE buffer was added,vortex and incubated at 800C for 25 minutes. Then eppendrof was vortexed and centrifuged at 12000x g for 5 minutes.Supernatant was flicked off and pellet was centrifuged again for 1 minute to remove the maximum supernatant with pipette. 50ul chloroform was then added and vortexed followed by 50ul NF water.then vortexed and centrifuged for 2 minute. After final centrifugation,debris was settled in the middle of two phases with extracted DNA in the upper phase and frozen at -200C to preserve for further analysis.

2. Extraction of fungal DNA:

Fungal DNA was extracted by Hoffman and Moan in 1988 by using salt precipitation method in a biosafety cabinet.

Materials:
Instruments Safety gloves

 Reagents

biosafety cabinet sterile tubes microcentrifuge vortex micropipettes autoclave uv -illuminator

Lysis buffer, TE buffer

Reagent preparation
50 ml Lysis buffer 1M Tris HCl EDTA Sodium dodecyl sulfate(SDS) B-mercaptoethanol Deionised sterile water 2.5ml 25ml 1.5g 0.5ml 20.5ml

Dissolved 1.5g of Sodium dodecyl sulfate(SDS) in deionised sterile water. Added 2.5ml of 1M Tris HCl,25ml of EDTA and 0.5ml B-mercaptoethanol.Then autoclaved and stored at room temprature. 1OX TE buffer(100mM) Trisma Base EDTA Water 1.21g 0.372g 100ml

Disolved 1.21g of Trisma base and 0.372g of EDTA in distilled water and adjusted the pH to 8.3 by adding concentrated HCl drop wise.Added distilled water to make the volume upto 100ml. Then autoclaved and stored at room temprature.

Methodology:
Sample were taken and centrifuged for 15 minutes at 14650 rpm. Supernatant was discarded and 300 ul of cell lysis solution to each microcentrifuge tube was added 1ul of RNase A to each tube as well. Suspended cells were incubated at 650C for overnight Samples were placed on ice for 10 minutes then 150ul of MPC protein precipitation reagent was added and vortexed mixed for 10 seconds. Pellet cellular debris by centrifugation in a microcentrifuge for 10 minutes at 14650 rpm. Supernatant was transferred to a clean microcentrifuge tube and added 500ul of isopropanal.Thoroughly mixed by inversion. DNA was centrifuged in a microcentrifuge for 10 minutes at 14650 rpm to get the pellet. Supernatant was removed by pipetting and discarded.Pellet was washed with 0.5ml of 70% ethanol.Ethanol was removed carefully by pipetting and discarded.DNA pellet was briefly centrifuge to remove any remaining ethanol. DNA was suspended in 35ul of TE buffer.Stored at 40C

DNA Purity testing:

Purity of DNA sample was tested with sepctrophotometer,by calculating ratio A260/A280. Pure DNA had a value of 1.6 to 1.8 impure DNA samples were re extracted by salt precipitation method.

3. Extraction from Plant leaf samples: Materials

Instruments Safetygloves biosafety cabinet sterile tubes

 Reagents

microcentrifuge vortex micropipettes autoclave uv -illuminator

CTAB buffer, EDTA, Distilled water

Reagent preparation
CTAB buffer 2%CTAB 20gmCTAB 20mM EDTA (0.5M) 100mM Tris Cl pH 8.0 100ml Tris-Cl (1M) 1.4M NaCl 280ml NaCl (5M) Make upto 1 liter with water,pH 7.5-8.0,and autoclave + 0.2%Mercaptoethanol Wash buffer 76%Ethanol 10mM NH4 Ac

Metodology:
Preheat 5ml CTAB (add 10l mercaptoethanol to each 5ml CTAB) in a bluetopped 50ml centrifuge tube at 60C. Remove and discard midribs, and wrap laminae in aluminium foil and freeze in liquid nitrogen. 0.5 1.0 gm tissue/5ml

CTAB.(Can store leaf material after liquid Nitrogen 1-2 days at 20 or 80 for longer periods) Gently crumble leaf tissue over cold pestle of liquid nitrogen. Grind frozen leaf with one spatula of fine sand add 0.5 spatula of PVPP powder after grinding. Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid leaving dry material around rim of tube. Adjust CTAB volume to give a slurrylike consistency, mix occasionally. Incubate for 60 min at 600 C Add equal volume of chloroform/iso-amyl alcohol (24:1), Mix for about 3min, then transfer contents to narrow bore centrifuge tubes. Balance by adding extra chlor/iso. Spin 5,000rpm for 10min (ensure correct tubes used), brake off. (For extra pure DNA isolation - spin and retain supernatant before chloroform extraction). Remove supernatant with wide-bore pastette (cut off blue tip) to clean tube, repeat chloroform extraction once. Supernatant should be clear, though may be coloured. Precipitate DNA with 0.66 vol. of cold isopropanol - can leave overnight. Spool out or spin down DNA, 2min at 2,000rpm. Transfer to 5ml wash buffer for 20min. Dry briefly and resuspend in 1ml T.E. (can be left overnight) Add 1l 10mg/ml RNAse to each 1ml T.E./DNA mixture and incubate for 60min at 370C. (If RNase in the sample doesn't matter stages 11 and 12 may be omitted) Dilute with 2 volumes TE and add 0.3vol 3M Sodium acetate (pH 8) + 2.5 vol cold 100% ethanol

Spool DNA out. Air dry and resuspend in 0.5 to 1ml TE or water (takes time) and freeze until required.

4. Extraction of Genomic DNA from whole blood Materials

Instruments Reagents Safety gloves, biosafety cabinet, sterile tubes, microcentrifuge, vortex, micropipettes, autoclave, uv -illuminator

Red blood cell lysis buffer,

Proteinase K buffer

Reagent preparation
Buffer A (Red blood cell lysis buffer)composition 0.32 M sucrose 10 mM Tris HCl

5 mM MgCl2 0.75% Triton-X-100 Adjust pH to 7.6

Buffer B (Proteinase K buffer) composition 20 mM Tris-HCl 4 mM Na2EDTA 100 mM NaCl Adjust pH to 7.4

Methodology:
Add 1 volume of buffer A to 1 volume of blood and 2 volumes of cold, sterile, distilled, deionised water. Vortex gently or invert tube 6-8 times and leave to incubate on ice for 2-3 minutes. Spin at 3500 rpm for 15 minutes at 4oC. Discard supernatant into 2.5% bleach solution and re-suspend pellet (vortex for 30 seconds at medium speed) in 2 ml of buffer A and 6 ml of water. Spin at 3500 rpm for 15 minutes at 4oC. The pellet should be white to cream in colour. If pellet is significantly red, repeat washing step again. Add 5 ml of Buffer B and 500 l of 10% SDS to pellet. Re-suspend pellet by vortexing vigorously for 30-60 seconds. Then add 50 l of Proteinase K solution (20mg/ml). The Proteinase K solution should be made fresh and refrigerated prior to use. Leave to incubate for two hours at 55oC in a water bath. Remove samples and leave to cool to room temperature (or leave for 2-3 minutes on ice). Add 4 ml of 5.3 M NaCl solution. Vortex gently for 15 seconds.

Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh tube. Take care not to dislodge pellet. Add an equal volume of cold isopropanol (stored at -20oC). Invert 5-6 times gently to precipitate DNA.

Remove DNA with a wide bore tip and transfer to a microfuge tube. Wash with 1 ml of 70% ethanol. Leave DNA to dry for 15-20 minutes at 37oC. Re-suspend in 300-400 l of Tris HCl, pH 8.5 (not TE!). Leave to re-dissolve overnight at room temperature. DNA can be safely refrigerated for up to a year. Long-term storage may involve ethanol at -70 0C.

5. DNA extraction of virus Materials

Instruments Safety gloves, Biosafety cabinet, sterile tubes, microcentrifuge, vortex, micropipettes, autoclave, uv -illuminator

Reagents Phenol chloroform, etanol or isopropanal, Tris or TE buffer

Metodology:

Break open (lyse) the cells or virus containing the DNA of interest.This is often done by sonicating or bead beating the sample.Vortexing with phenol (sometimes heated) is often effective for breaking down protienacious cellular walls or viral capsids. The addition of a detergent such as SDS is often necessary to remove lipid membranes.

DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases.

DNA is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added.

Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet.

After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE.

Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light.

DNA Purity testing:

Purity of DNA sample was tested with sepctrophotometer,by calculating ratio A260/A280. Pure DNA had a value of 1.6 to 1.8 impure DNA samples were re extracted by salt precipitation method.

Agarose gel electrophoresis:

The detection of extracted DNA on 1.5% agarose gel electrophoresis. The results were analyzed by UV-illuminator system.

Reagent preparation
TBE Buffer 10X Trisma base Boric acid EDTA Distilled water 108g 55g 9.3g upto 1L

Dissolved reagents and adjusted pH to 8.0 with HCl. Shaking was done vigorously with a stirrer; added water to make a final volume of 1liter.Autoclaved and stored at room temprature. Ethidium bromide (10 mg/ml) 0.2g of ethidium bromide was dissolved in distilled water and volume was made upto 20ml. stored the solution at room temprature.

Methodology:
1.5g of agarose was dissolved in 100ml of TBE(10X)buffer by heating in an oven for 2 minutes to make a homogeneous gel The solution was handeled with gloves and was allowed to cool approximately to 500c. few drops of ethidium bromide were added and shaken. Whn cooled it was pouerd into a comb placed gel tray and allowed to splidify for almost half hour. After the soldification of gel,the comb was carefully removed and bubbles were also removed if any .

20ul of DNA sample was mixed with 4ul of tracking dye and loaded in a well. Electrophoresis was done at a voltage of 120V for approximately 20 minutes. The gel was photographed by UV-illuminator. A sharp band was indicated that the sample was not contaminated and determined the purity of DNA sample

DNA Purity testing:

Purity of DNA sample was tested with sepctrophotometer,by calculating ratio A260/A280. Pure DNA had a value of 1.6 to 1.8 impure DNA samples were re extracted by salt precipitation method.

Spectrophotometer:
Spectrophotometer is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It deals with visible light,near ultraviolet,and near infrared,but does not cover time resolved spectroscopic techniques. Spectroscopy involves the use of a spectrophotometer. A spectrophotometer is a photometer that can measure intensity as a function of the light source wavelenght. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflection measurement.