The document describes the alkaline lysis method for plasmid purification from bacterial cells. It involves four main steps: 1) resuspending harvested cells, 2) lysing the cells with NaOH and SDS, 3) neutralizing with potassium acetate, and 4) clearing the lysate by centrifugation. This separates plasmid DNA from genomic DNA and other cell debris. The purified plasmid DNA can then be analyzed using agarose gel electrophoresis.

1. PLASMID ISOLATIONANDPURIFICATION

2. PRINCIPLEPlasmid for routine molecular cloning method is often purified by one of the following methods :-Alkaline lysis methodBoiling methodLithium chloride base 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.Sunday, April 04, 20102HIMANSHU CHAUDHRY

3. ALKALINE LYSIS methodAlkaline lysis method is one of the most commonly used method for lysis bacterial cells prior to plasmid purification. It has four basic steps :- 1. Resuspension : Harvested bacterial cells are resuspended by using solution I contains EDTA (ethylene diaminetetra-acetic acid) and Tris-CL. EDTA – chelates the magnesium and calcium ions Tris-CL – maintains pH. 2 . LYSIS : Cells are lysis with alkaline solution II contains NaOH and SDS (sodium dodecyl sulfate).NaOH -- denatures the chromosomal and plasmid DNAs as well as proteins. SDS -- solubilizes the phospholipids and protein components of the cell membrane, leading to lysis and release of the cell membrane.3. NEUTRALIZATION : The lysate is neutralized by addition of solution III of acidic potassium acetate. The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate and denatured proteins, chromosomal DNA and cellular debris are co-precipitated in insoluble.. 4. CLEARING OF LYSATES : Precipitated debris is removed by either high speed centrifugation or filtration, producing cleared lysatesSunday, April 04, 20103 HIMANSHU CHAUDHRY

4. continue…. Step ‘s and procedure in ALKALINE LYSIS METHODSunday, April 04, 20104 HIMANSHU CHAUDHRY

5. .Continue…..Harvest cells by centrifugationSpin ~5,000 rcfSupernatant (clear)‏E. coli culture(cloudy)‏Pelleted cellsDiscard supernatant Residual media may interfere with downstream stepsResuspend cells in buffer Thoroughly resuspend cells, making sure that no clumps remain. P1 buffer contains: •Tris-Cl (buffering agent)‏ •EDTA (metal chelator)‏ •RNase A (degrades RNA)‏

6. Continue….Sunday, April 04, 20106Lyse cells with SDS/NaOH solutionAdding buffer P2 causes solution to become viscous 1. Sodium dodecylsulfate• Dissolves membranes• Binds to and denatures proteins 2. NaOH• Denatures DNABecause plasmids are supercoiled, both DNA strands remain entangled after denaturation HIMANSHU CHAUDHRY

7. sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS)‏ (H2O sol. = 10%) (H2O sol. < 0.02%)‏Continue…Neutralize NaOH with potassium acetate solutionMixing with buffer N3 causes a fluffy white precipitate to form. 1. Potassium acetate / acetic acid solution • Neutralizes NaOH (renatures plasmid DNA)‏ • Converts soluble SDS to insoluble PDS 2. Guanidine hydrochloride (GuCl) • Chaotropic salt; facilitates DNA binding to silica in later stepsSunday, April 04, 20107 HIMANSHU CHAUDHRY

8. Continue….Sunday, April 04, 20108Separate plasmid DNA from contaminants by centrifugationSupernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA HIMANSHU CHAUDHRY

9. Sunday, April 04, 20109Add cleared lysate to column and centrifugeThe high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the columnCentrifugeNucleic acidsSilica-gel membraneFlow through(discard)‏ HIMANSHU CHAUDHRY

10. Sunday, April 04, 201010Wash the silica membrane to remove residual contaminantsBuffer PB contains isopropanol and GuClCentrifugePB bufferNucleic acidsNucleic acidsPB + contaminantsBuffer PE contains ethanol and Tris-ClCentrifugePE bufferNucleic acidsNucleic acidsPE + contaminants (including residual GuCl)‏HIMANSHU CHAUDHRY

11. Sunday, April 04, 201011Elute purified DNA from the columnBuffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible EtOH contamination (from residual PE buffer)‏EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O may also be used.CentrifugeEB bufferNucleic acidsEB + DNA HIMANSHU CHAUDHRY

12. Continue…. PLASMID PREPARATION 1.5ml of bacterial culture was taken in centrifuge tube ↓ Centrifuge of bacterial culture at 13,000 rpm ∕ 30 seconds ↓ Collection of pellet in fresh eppendorf’s tubes ↓ Addition of 100µl S1 buffer to the pellet ↓ Addition of 200µl S2 buffer and mixing of the sample by inverting 6-8 times ↓ Addition of 150µl of S3 buffer and mixing of the sample by inverting 6-8 times ↓ Addition of 450µl of P1 buffer and mixing of the sample by inverting 6-8 times ↓ Centrifugation at 13,000 rpm ∕ 30 sec ↓ Collection of supernatant in a fresh tube ↓ Addition of 20µl DBM into the supernatant and mixing the sample by inverting 6-8 timesSunday, April 04, 201012 HIMANSHU CHAUDHRY

13. Continue... Incubation at room temperature for 1 minute ↓ Centrifugation at 13,000 rpm ∕ 30 sec ↓ Removal of supernatant ↓ Addition of 500µl wash buffer to the pellet ↓ Centrifugation at 13,000 rpm / 30 sec↓ Centrifugation until complete removal of wash buffer ↓ Addition of 20µl Elution buffer to the pellet ↓ Incubation at room temperature for 1 minute ↓ Centrifugation at 10,000 rpm/30 second ↓ Collection of Elutein a fresh tube↓ store in −20°C in freezeSunday, April 04, 201013 HIMANSHU CHAUDHRY

14. Gel electrophoresisAgarose 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.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.Sunday, April 04, 201014 HIMANSHU CHAUDHRY

15. PREP. Of 1% AGAROSE GELTake 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.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.Sunday, April 04, 201015 HIMANSHU CHAUDHRY

16. Continue….Most Agarose gels:1. 1% gels are common for many applications.2. 0.7%: good separation or resolution of large 5–10kb DNA fragments3. 2% good resolution for small 0.2–1kb fragments.4. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case.Sunday, April 04, 201016 HIMANSHU CHAUDHRY

17. Continue…BufferThe most common buffers for Agarose gel:TAE: Tris acetate EDTATBE: Tris/Borate/EDTASB: Sodium borate.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.Sunday, April 04, 2010 HIMANSHU CHAUDHRY17

18. Continue…..An agarose gel is prepared by combining agarose powder and a buffer solution.Sunday, April 04, 2010HIMANSHU CHAUDHRY18BufferFlask for boilingAgarose 

19. Sunday, April 04, 2010 HIMANSHU CHAUDHRY19Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.Buffer solution Agarose Powder

20. Melting the Agarose Agarose is insoluble at room temperature The agarose solution is boiled until clearSunday, April 04, 2010HIMANSHU CHAUDHRY20

21. Gel casting tray & combsSunday, April 04, 2010 HIMANSHU CHAUDHRY21

22. Preparing the Casting TraySunday, April 04, 2010 HIMANSHU CHAUDHRY22Seal 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.

23. POURING THE GEL IN TRAYSunday, April 04, 2010 HIMANSHU CHAUDHRY23Allow the agarose solution to cool slightly (~60°C) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

24. Each of the gel combs should be submerged in the melted agarose solution. Sunday, April 04, 2010 HIMANSHU CHAUDHRY24

25. Sunday, April 04, 2010 HIMANSHU CHAUDHRY25When 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.

26. Sunday, April 04, 2010HIMANSHU CHAUDHRY26 Place the gel in the electrophoresis chamber.

27. Sunday, April 04, 2010 HIMANSHU CHAUDHRY27DNABUFFERWELLSANODE (positive)CATHODE(Negative)Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

28. Sample Preparation6X Loading Buffer:   Bromophenol Blue (for color)  Glycerol (for weight)Sunday, April 04, 2010 HIMANSHU CHAUDHRY28Mix 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.

29. Loading the GelSunday, April 04, 2010 HIMANSHU CHAUDHRY29Carefully 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.

30. Running the GelSunday, April 04, 2010 HIMANSHU CHAUDHRY30Place 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.

31. Sunday, April 04, 2010 HIMANSHU CHAUDHRY31Cathode(-)DNA(-) wells Bromophenol BlueAnode(+)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.

32. Observe the gel under UV LightSunday, April 04, 2010 HIMANSHU CHAUDHRY32Assessing your plasmid preparation1. Quantify abundance (A260) and purity (A260/A280)‏2. Verify by restriction digestion3. Run undigested plasmid to see if it is mostlysupercoiled←SUPERCOILED←DENATURED

33. ENDPRESENTED BY:-HIMANSHU CHAUDHRY