plasmid isolation or purification by alkalinelysis method.

1. PLASMID ISOLATIONANDPURIFICATION

2. PRINCIPLE Plasmid for routine molecular cloning method is often purified by one of the following methods :- Alkaline lysis method Boiling method Lithium 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, 2010 2 HIMANSHU CHAUDHRY

3. ALKALINE LYSIS method Alkaline 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 lysates Sunday, April 04, 2010 3 HIMANSHU CHAUDHRY

4. continue…. Step ‘s and procedure in ALKALINE LYSIS METHOD Sunday, April 04, 2010 4 HIMANSHU CHAUDHRY

5. . Continue….. Harvest cells by centrifugation Spin ~5,000 rcf Supernatant (clear)‏ E. coli culture (cloudy)‏ Pelleted cells Discard supernatant Residual media may interfere with downstream steps Resuspend 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, 2010 6 Lyse cells with SDS/NaOH solution Adding buffer P2 causes solution to become viscous 1. Sodium dodecylsulfate • Dissolves membranes • Binds to and denatures proteins 2. NaOH • Denatures DNA Because 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 solution Mixing 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 steps Sunday, April 04, 2010 7 HIMANSHU CHAUDHRY

8. Continue…. Sunday, April 04, 2010 8 Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA HIMANSHU CHAUDHRY

9. Sunday, April 04, 2010 9 Add cleared lysate to column and centrifuge The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column Centrifuge Nucleic acids Silica-gel membrane Flow through (discard)‏ HIMANSHU CHAUDHRY

10. Sunday, April 04, 2010 10 Wash the silica membrane to remove residual contaminants Buffer PB contains isopropanol and GuCl Centrifuge PB buffer Nucleic acids Nucleic acids PB + contaminants Buffer PE contains ethanol and Tris-Cl Centrifuge PE buffer Nucleic acids Nucleic acids PE + contaminants (including residual GuCl)‏ HIMANSHU CHAUDHRY

11. Sunday, April 04, 2010 11 Elute purified DNA from the column Buffer 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. Centrifuge EB buffer Nucleic acids EB + 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 times Sunday, April 04, 2010 12 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 freeze Sunday, April 04, 2010 13 HIMANSHU CHAUDHRY

14. Gel electrophoresis 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. 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, 2010 14 HIMANSHU CHAUDHRY

15. PREP. Of 1% AGAROSE GEL 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. 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, 2010 15 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 fragments 3. 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, 2010 16 HIMANSHU CHAUDHRY

17. Continue… Buffer The most common buffers for Agarose gel: TAE: Tris acetate EDTA TBE: Tris/Borate/EDTA SB: 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 CHAUDHRY 17

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

19. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 19 Combine 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 clear Sunday, April 04, 2010 HIMANSHU CHAUDHRY 20

21. Gel casting tray & combs Sunday, April 04, 2010 HIMANSHU CHAUDHRY 21

22. Preparing the Casting Tray Sunday, April 04, 2010 HIMANSHU CHAUDHRY 22 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.

23. POURING THE GEL IN TRAY Sunday, April 04, 2010 HIMANSHU CHAUDHRY 23 Allow 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 CHAUDHRY 24

25. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 25 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.

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

27. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 27 DNA BUFFER WELLS ANODE (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 Preparation 6X Loading Buffer:   Bromophenol Blue (for color)  Glycerol (for weight) Sunday, April 04, 2010 HIMANSHU CHAUDHRY 28 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.

29. Loading the Gel Sunday, April 04, 2010 HIMANSHU CHAUDHRY 29 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.

30. Running the Gel Sunday, April 04, 2010 HIMANSHU CHAUDHRY 30 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.

31. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 31 Cathode (-) DNA (-)   wells  Bromophenol Blue Anode (+) 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 Light Sunday, April 04, 2010 HIMANSHU CHAUDHRY 32 Assessing your plasmid preparation 1. Quantify abundance (A260) and purity (A260/A280)‏ 2. Verify by restriction digestion 3. Run undigested plasmid to see if it is mostly supercoiled ←SUPERCOILED ←DENATURED

33. END PRESENTED BY:- HIMANSHU CHAUDHRY