Himanshu chaudhry


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plasmid isolation or purification by alkalinelysis method.

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Himanshu chaudhry

  2. 2. PRINCIPLE<br />Plasmid for routine molecular cloning method is often purified by one of the following methods :-<br />Alkaline lysis method<br />Boiling method<br />Lithium chloride base <br /> The purity of plasmid isolated by these above methods depends on how efficiently a method can separate plasmid DNA from genomic DNA. Most of these plasmid purification methods allow the preferential recovery of circular plasmid DNA over linear chromosomal DNA.<br />Sunday, April 04, 2010<br />2<br />HIMANSHU CHAUDHRY<br />
  3. 3. ALKALINE LYSIS method<br />Alkaline lysis method is one of the most commonly used method for lysis bacterial cells prior to plasmid purification. It has four basic steps :-<br /> 1. Resuspension : Harvested bacterial cells are resuspended by using solution I contains<br /> EDTA (ethylene diaminetetra-acetic acid) and Tris-CL.<br /> EDTA – chelates the magnesium and calcium ions <br /> Tris-CL – maintains pH. <br />2 . LYSIS : Cells are lysis with alkaline solution II contains NaOH and SDS (sodium dodecyl sulfate).<br />NaOH -- denatures the chromosomal and plasmid DNAs as well as proteins.<br /> SDS -- solubilizes the phospholipids and protein components of the cell membrane, leading to lysis <br /> and release of the cell membrane.<br />3. NEUTRALIZATION : The lysate is neutralized by addition of solution III of acidic potassium acetate. The high salt<br /> concentration causes potassium dodecyl sulfate (KDS) to precipitate and denatured proteins, <br /> chromosomal DNA and cellular debris are co-precipitated in insoluble.<br />.<br /> 4. CLEARING OF LYSATES : Precipitated debris is removed by either high speed centrifugation or filtration, producing cleared lysates<br />Sunday, April 04, 2010<br />3<br /> HIMANSHU CHAUDHRY<br />
  4. 4. continue….<br /> Step ‘s<br /> and<br /> procedure<br /> in<br /> ALKALINE LYSIS METHOD<br />Sunday, April 04, 2010<br />4<br /> HIMANSHU CHAUDHRY<br />
  5. 5. .<br />Continue…..<br />Harvest cells by centrifugation<br />Spin ~5,000 rcf<br />Supernatant (clear)‏<br />E. coli culture<br />(cloudy)‏<br />Pelleted cells<br />Discard supernatant<br /> Residual media may interfere with downstream steps<br />Resuspend cells in buffer<br /> Thoroughly resuspend cells, making sure that no <br /> clumps remain. P1 buffer contains:<br /> •Tris-Cl (buffering agent)‏<br /> •EDTA (metal chelator)‏<br /> •RNase A (degrades RNA)‏<br />
  6. 6. Continue….<br />Sunday, April 04, 2010<br />6<br />Lyse cells with SDS/NaOH solution<br />Adding buffer P2 causes solution to become viscous<br /> 1. Sodium dodecylsulfate<br />• Dissolves membranes<br />• Binds to and denatures proteins<br /> 2. NaOH<br />• Denatures DNA<br />Because plasmids are supercoiled, both DNA strands remain entangled after denaturation<br /> HIMANSHU CHAUDHRY<br />
  7. 7. sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS)‏<br /> (H2O sol. = 10%) (H2O sol. < 0.02%)‏<br />Continue…<br />Neutralize NaOH with potassium acetate solution<br />Mixing with buffer N3 causes a fluffy white precipitate to form.<br /> 1. Potassium acetate / acetic acid solution<br /> • Neutralizes NaOH (renatures plasmid DNA)‏<br /> • Converts soluble SDS to insoluble PDS<br /> 2. Guanidine hydrochloride (GuCl)<br /> • Chaotropic salt; facilitates DNA binding to silica in <br /> later steps<br />Sunday, April 04, 2010<br />7<br /> HIMANSHU CHAUDHRY<br />
  8. 8. Continue….<br />Sunday, April 04, 2010<br />8<br />Separate plasmid DNA from contaminants by centrifugation<br />Supernatant contains:<br /> - Plasmid DNA<br /> - Soluble cellular constituents<br /> Pellet contains:<br /> - PDS<br /> - Lipids<br /> - Proteins<br /> - Chromosomal DNA<br /> HIMANSHU CHAUDHRY<br />
  9. 9. Sunday, April 04, 2010<br />9<br />Add cleared lysate to column and centrifuge<br />The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column<br />Centrifuge<br />Nucleic acids<br />Silica-gel membrane<br />Flow through<br />(discard)‏<br /> HIMANSHU CHAUDHRY<br />
  10. 10. Sunday, April 04, 2010<br />10<br />Wash the silica membrane to remove residual contaminants<br />Buffer PB contains isopropanol and GuCl<br />Centrifuge<br />PB buffer<br />Nucleic acids<br />Nucleic acids<br />PB + contaminants<br />Buffer PE contains ethanol and Tris-Cl<br />Centrifuge<br />PE buffer<br />Nucleic acids<br />Nucleic acids<br />PE + contaminants <br />(including residual GuCl)‏<br />HIMANSHU CHAUDHRY<br />
  11. 11. Sunday, April 04, 2010<br />11<br />Elute purified DNA from the column<br />Buffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible EtOH contamination (from residual PE buffer)‏<br />EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O may also be used.<br />Centrifuge<br />EB buffer<br />Nucleic acids<br />EB + DNA<br /> HIMANSHU CHAUDHRY<br />
  12. 12. Continue….<br /> PLASMID PREPARATION<br /> 1.5ml of bacterial culture was taken in centrifuge tube<br /> ↓<br /> Centrifuge of bacterial culture at 13,000 rpm ∕ 30 seconds<br /> ↓ <br /> Collection of pellet in fresh eppendorf’s tubes<br /> ↓<br /> Addition of 100µl S1 buffer to the pellet<br /> ↓ <br /> Addition of 200µl S2 buffer and mixing of the sample by inverting 6-8 times<br /> ↓ <br /> Addition of 150µl of S3 buffer and mixing of the sample by inverting 6-8 times<br /> ↓<br /> Addition of 450µl of P1 buffer and mixing of the sample by inverting 6-8 times <br /> ↓<br /> Centrifugation at 13,000 rpm ∕ 30 sec<br /> ↓<br /> Collection of supernatant in a fresh tube<br /> ↓ <br /> Addition of 20µl DBM into the supernatant and mixing the sample by inverting 6-8 times<br />Sunday, April 04, 2010<br />12<br /> HIMANSHU CHAUDHRY<br />
  13. 13. Continue...<br /> Incubation at room temperature for 1 minute<br /> ↓<br /> Centrifugation at 13,000 rpm ∕ 30 sec<br /> ↓<br /> Removal of supernatant<br /> ↓ <br /> Addition of 500µl wash buffer to the pellet<br /> ↓<br /> Centrifugation at 13,000 rpm / 30 sec<br />↓<br /> Centrifugation until complete removal of wash buffer<br /> ↓<br /> Addition of 20µl Elution buffer to the pellet <br />↓<br /> Incubation at room temperature for 1 minute<br /> ↓<br /> Centrifugation at 10,000 rpm/30 second<br /> ↓<br /> Collection of Elutein a fresh tube<br />↓<br /> store in −20°C in freeze<br />Sunday, April 04, 2010<br />13<br /> HIMANSHU CHAUDHRY<br />
  14. 14. Gel electrophoresis<br />Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape. The purpose of the gel will be either to visualize the DNA,to quantify it or to isolate a particular band. Agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole.<br />DNA is visualized in the gel by addition of ethidium bromide , binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light.<br />Sunday, April 04, 2010<br />14<br /> HIMANSHU CHAUDHRY<br />
  15. 15. PREP. Of 1% AGAROSE GEL<br />Take one 250 mg Agarose tablet in 25 ml of 1x TAE buffer. The tablet gets disintegrated with in 1 min. Mix the content and heat it in a microwave for 30 seconds.<br />Mix the content and allow the Agarose to cool to 50°C and pour it in a plate that was sealed on either sides using a tape. After the gel is solidified, remove the tape and use it for electrophoresis of DNA samples.<br />Sunday, April 04, 2010<br />15<br /> HIMANSHU CHAUDHRY<br />
  16. 16. Continue….<br />Most Agarose gels:<br />1. 1% gels are common for many applications.<br />2. 0.7%: good separation or resolution of large 5–10kb DNA fragments<br />3. 2% good resolution for small 0.2–1kb fragments.<br />4. Up to 3% can be used for separating very tiny fragments but a <br /> vertical polyacrylamide gel is more appropriate in this case.<br />Sunday, April 04, 2010<br />16<br /> HIMANSHU CHAUDHRY<br />
  17. 17. Continue…<br />Buffer<br />The most common buffers for Agarose gel:<br />TAE: Tris acetate EDTA<br />TBE: Tris/Borate/EDTA<br />SB: Sodium borate.<br />TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />17<br />
  18. 18. Continue…..<br />An agarose gel is prepared by combining agarose powder and a buffer solution.<br />Sunday, April 04, 2010<br />HIMANSHU CHAUDHRY<br />18<br />Buffer<br />Flask for boiling<br />Agarose <br />
  19. 19. Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />19<br />Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.<br />Buffer solution<br /> Agarose Powder<br />
  20. 20. Melting the Agarose <br />Agarose is insoluble at room temperature <br />The agarose solution is boiled until clear<br />Sunday, April 04, 2010<br />HIMANSHU CHAUDHRY<br />20<br />
  21. 21. Gel casting tray & combs<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />21<br />
  22. 22. Preparing the Casting Tray<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />22<br />Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.<br />
  23. 23. POURING THE GEL IN TRAY<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />23<br />Allow the agarose solution to cool slightly (~60°C) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.<br />
  24. 24. Each of the gel combs should be submerged in the melted agarose solution. <br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />24<br />
  25. 25. Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />25<br />When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape. <br />
  26. 26. Sunday, April 04, 2010<br />HIMANSHU CHAUDHRY<br />26<br /> Place the gel in the electrophoresis chamber.<br />
  27. 27. Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />27<br />DNA<br />BUFFER<br />WELLS<br />ANODE (positive)<br />CATHODE<br />(Negative)<br />Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.<br />
  28. 28. Sample Preparation<br />6X Loading Buffer: <br />  Bromophenol Blue (for color)<br />  Glycerol (for weight)<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />28<br />Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.<br />
  29. 29. Loading the Gel<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />29<br />Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.<br />
  30. 30. Running the Gel<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />30<br />Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.<br />
  31. 31. Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />31<br />Cathode<br />(-)<br />DNA<br />(-)<br /><br /> wells<br /> Bromophenol Blue<br />Anode<br />(+)<br />After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.<br />
  32. 32. Observe the gel under UV Light<br />Sunday, April 04, 2010<br /> HIMANSHU CHAUDHRY<br />32<br />Assessing your plasmid preparation<br />1. Quantify abundance (A260) and purity (A260/A280)‏<br />2. Verify by restriction digestion<br />3. Run undigested plasmid to see if it is mostly<br />supercoiled<br />←SUPERCOILED<br />←DENATURED<br />
  33. 33. END<br />PRESENTED BY:-<br />HIMANSHU CHAUDHRY<br />