Genomic Dna Isolation From Blood, Bacteria and Plasmid DNA Isolation

Genomic Dna Isolation From Blood, Bacteria and Plasmid DNA Isolation

• 2.
  • Consumables Required •Equipments Required • Reagents Required • Protocol • Analysis
• 3.
  Consumables Required
• 4.
  Equipments/tools Required
• 5.
  Reagents Required To bekept at room temperature : • TRIS Base • NaCl • EDTA • KHCO3 • NH4Cl • CH3COONa • SDS 20% • Absolute ethanol • Phenol • Chloroform • Iso amylalcohol To be stored at 4°C : Proteinase K
• 6.
  PREPARATION OF WORKINGSOLUTIONS 1) Lysis buffer (1000 ml) NH4Cl 155 ml KHCO3 10 ml EDTA 0.2 ml H2O 834.8 ml 2) Digestion (SE) buffer (1000 ml) NaCl 75 ml TRIS 10 ml EDTA 4 ml H2O 911 ml
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  LYSIS CYCLE • Takeblood in oakridge tube • Add 2×lysis buffer • Incubate in ice for 10 minutes • Centrifuge 10,000 RPM at 4OC for 10 minutes • Discard the supernatant, WBC pellet will be left • Add 5 ml SE buffer and dissolve WBC pellet • Add SDS buffer (125 ml of 20% SDS) • Add proteinase K (25 ml) • Overnight incubate at 65OC
• 8.
  PHENOL EXTRACTION • Addequal amount of phenol at pH 8 • Mix well for 5 minutes • Centrifuge at 12,000 RPM at 280C for 10 minutes • Collect the upper aqueous phase in oakridge tube • Add equal amount of P:C:I • Mix well for 10 minutes • Centrifuge at 12,000 RPM at 280C for 10 minutes • Again collect the upper aqueous phase in another oakridge tube • Add equal amount of C:I (~5 ml) • Mix well 5-10 minutes • Centrifuge at 12,000 RPM at 280C for 5-10 minutes
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  DNA PRECIPITATION • Seperateaqueous phase in borosil tube • Add 0.1 volume of CH3COONa • Add 2 volume of chilled ethanol • Mix gently and white strands of DNA will appear
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  WASHING OF DNA •Spool out DNA and transfer in eppendorf tube • Add 1 ml of 70% ethanol, mix gently and leave for 1-2 min • Centrifuge at 10,000 RPM at 25oC for 5 minutes • Decant the 70% ethanol • Repeat above 2 steps • Leave eppendorf open and allow the DNA pellet to dry
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  DISSOLUTION OF DNA •Add 500 ul of TE buffer to dissolve the dry pellet present in the eppendorf • Close the lid of eppendorf and cover it with parafilm • Keep at 650C for 10-15 min • Store in -200C
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  Bacterial Genomic DNAIsolation
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  Materials Required • LBBroth • E. coli DH5αcells • TE buffer (pH 8.0) • 10% SDS • Proteinase K • Phenol-chloroform mixture • 5M Sodium Acetate (pH 5.2) • Isopropanol • 70% ethanol
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  Preparation of Reagents 1.TE BUFFER (pH 8.0):10 mm Tris HCl (pH 8.0), 1 mm EDTA (pH 8.0) 2. 10% SDS:Dissolve 10 g of SDS in 100 ml autoclaved distilled water. 3. PROTEINASE K:Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water. 4. PHENOL – CHLOROFORM MIXTURE: Mix equal volume of phenol with chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for 15 minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it on ice. 5. 5M SODIUM ACETATE:Dissolve 41 g of sodium acetate in 100 ml distilled water and adjust pH withdilute acetic acid (pH 5.2).
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  PROCEDURE 1. 2 mlovernight culture is taken and the cells are harvested by centrifugation for 10 minutes 2. 875 µl of TE buffer is added to the cell pellet and the cells are resuspended in the buffer by gentle mixing. 3. 100 µl of 10% SDS and 5 µl of Proteinase K are added to the cells. 4. The above mixture is mixed well and incubated at 370 C for an hour in an incubator. 5. 1 ml of phenol-chloroform mixture is added to the contents, mixed well by inverting and incubated at room temperature for 5 minutes. 6. The contents are centrifuged at 10,000 rpm for 10 minutes at 40 C. 7. The highly viscous jelly like supernatant is collected using cut tips and is transferred to a fresh tube.
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  8. The processis repeated once again with phenol-chloroform mixture and the supernatant is collected in a fresh tube. 9. 100 µl of 5M sodium acetate is added to the contents and is mixed gently. 10. 2 ml of isopropanol is added and mixed gently by inversion till white strands of DNA precipitates out. 11. The contents are centrifuged at 5,000 rpm for 10 minutes. 12. The supernatant is removed and 1ml 70% ethanol is added. 13. The above contents are centrifuged at 5,000 rpm for 10 minutes. 14. After air drying for 5 minutes 200 µlof TE buffer or distilled water is added. 15. 10 µl of DNA sample is taken and is diluted to 1 or 2 ml with distilled water. 16. The concentration of DNA is determined using a spectrophotometer at 260/280 nm.
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  Plasmid Isolation protocol
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  When bacteria arelysed under alkaline conditions both DNA and proteins are precipitated. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.
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  Materials Required 1. LuriaBroth 2. Bacterial cells containing plasmid 3. TE buffer(pH 8.0) 4. Solution I 5. Solution II 6. Solution III 7. Phenol-chloroform mixture 8. Isopropanol 9. 70% ethanol 10. Autoclaved Distilled Water
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  Preparation of Reagents SolutionI (Lysis buffer I) 10ml 500mM Glucose 2ml 500mM EDTA pH 8.0 2.5ml 1M Tris pH 8.0 85.5ml H2O Autoclave and store at 4°C Solution II (Lysis buffer II) Freshly prepared 0.2 N NaOH 1% SDS. Store at RT Solution III (Lysis buffer III) pH 6 60ml 5M potassium acetate (49.07g potassium acetate in 100ml H2O) 11.5ml glacial acetate 28.5ml H2O
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  PROCEDURE • Take 2ml overnight culture and harvest cells by centrifugation for 5 minutes. Discard the supernatant carefully. • Add 100 µl of solution I to the cell pellet and resuspend the cells by gentle mixing. • Incubate the above mixture at room temperature for 5 minutes. • Add 200 µl of solution II to the mixture and mix by inverting the tubes for 5 minutes. • Incubate for 5-10 minutes at room temperature. • Add 500µl of ice cold solution III tothe mixture and mix by inverting the tube. • Incubate on ice for 10 minutes. • Centrifuge at 10,000 rpm for 5 minutes.
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  • Transfer thesupernatant into fresh tube. • Add 400 µl of phenol-chloroform mixture to the contents, mix well by inverting and incubate them at room temperature for 5 minutes. • Centrifuge at 10000 rpm for 5 minutes. • Collect the supernatant (viscous) using cut tips and transfer to a fresh tube. • Add 0.8 ml of isopropanol and mix gently by inversion. Incubate for 30 min at room temperature. • Centrifuge the contents at 10,000 rpm for 10 minutes • Discard the supernatant after centrifugation. • After air drying for 5 minutes, add 100 µl of TE buffer or autoclaved distilled water to the pellet to resuspend the plasmid DNA.
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  • The contaminatedsalt in the DNA pellet can be removed with 70% ethanol washing. • Take 10 µl of plasmid sample and dilute to 1 ml withdistilled water for spectrometric analysis. • The concentration of plasmid is determined using a spectrophotometer at 260/280 nm. • An aliquot of plasmid DNA is used for agarose electrophoresis for quantitative and qualitative analyses.
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  CHECKING THE EXTRACTED DNA
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  1. Electrophoresis inagarose gels- qualitative VPB 321
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  2. Espectrophotometer/ nanometer-quantitative -Gives 260/ 280 ratio which tells us about purity of DNA. 260/280 = 1.8 stable 260/280 = 2.0 > (contaminated with proteins) 260/280 = 1.6 < (contaminated with RNA) -Also it quantifies and displays concentration of DNA in ng/ulves the 260/ 280 ratio which tells us about purity of DNA. 260/280 = 1.8 stable 260/280 = 2.0 > (contaminated with proteins) 260/280 = 1.6 < (contaminated with RNA)  Also it quantifies and displays concentration of DNA in ng/ul. VPB 321