1. Rapid isolation of plasmid DNA by boiling lysis Aim: To extract plasmid from bacteria using boiling lysis method.

Principle: Manipulation of plasmid for genetic engineering requires that they should be obtained in significant amounts, with desirable purity. To isolate plasmid DNA from host cells chemical or heat treatment methods have been used. This method yields a crude preparation of plasmid DNA from Escherichia coli or similar bacteria. In boiling lysis method, sudden temperature gradient is used to disrupt the cells and separate the plamid DNA from the genomic DNA which forms part of the pellet along with cell debris. Lysozyme and STET buffer are useful in lysis of the cells. The plasmid, present in supernatant is precipitated using isopropanol (Refer to section 3.5 of Working with DNA). The extracted plasmid could be stored in TE buffer that maintains the integrity of the DNA. The protocol is fast and easy, and does not involve phenol-chloroform extraction. Hence the resultant plasmid preparation is not free of cellular proteins and RNA and thus needs to be cleaned up before use for sequencing or similar applications that require highly pure DNA. For pure plasmid phenol chloroform extraction can also be carried out. Materials Required: Glassware: Test tube, measuring cylinder, thermometer. Plastic ware: 1.5 ml Microfuge tube, Test tube stand, Float, Micro pipettes (1 ml, 200l, 20l). Reagents: LB broth, Antibiotic (in required concentration) STET (Sucrose-Tris-EDTA-Triton) buffer (8% sucrose, 50mM Tris-HCl pH 8.1, 50mM EDTA, 5% Triton X-100. To make one liter of STET buffer, add 80g sucrose and 50ml Triton X-100 to ~700ml of water and autoclave. Then add 100ml of 0.5M EDTA and 25ml of 2M Tris-HCl pH 8.1, and bring up the volume to 1000ml with autoclaved water in a sterile flask). Lysozyme (Stock: 10mg/ml in 100mM Tris-HCl pH 8.1) 0.6M Sodium acetate (diluted from a 3.0M stock) Isoopropanol 70% Ethanol TE (1 mM EDTA, 10mM Tris-Cl)

Optional (For phenol chloroform extraction) : 1. Phenol:Chloroform:Isoamyl alcohol (25:24:1) 2. Chloroform:Isoamyl alcohol (24:1)

Misc: Toothpicks, shaking incubator (at 37 C), table top centrifuge machine, water bath(95C), crushed ice.
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Protocol: 1. Pick a single colony of the desired bacterial strain (with the plasmid) and inoculate into 2-4ml of LB broth. Incubate the culture tube at 37C with vigorous shaking overnight (10-12 hours).

2. Fill a 1.5ml microfuge tube with the bacterial culture, and spin about 30s to
pellet the cells. Discard the supernatant.

(Optional step: You may pellet another 1.5ml of culture in the same tube if you want more plasmid.)
3. Resuspend the pellet in 210l of STET+lysozyme solution (per 200l of STET, add 10l of lysozyme stock). Mix well with the pipette tip so that the entire pellet is dissolved. 4. Vortex the tube briefly and incubate it at room temperature for 2-4 min. 5. Heat for 2 min in a 95C block.

(Do not exceed this time, else DNA yield may be affected) 6. Add 200l of 0.6M NaOAc and mix the contents of the tube well by inverting several times. (Do not vortex at this stage since it may lead to shearing of the genomic DNA that may subsequently contaminate the plasmid preparation.)
7. Spin for 6-8 min in a microcentrifuge at room temperature at maximum speed. You should get a fluffy white pellet.

(On a standard tabletop centrifuge, the speed is 13,000 rpm.)

8. Using a toothpick, carefully remove pellet from the tube; now discard the toothpick, along with the pellet attached to it. 9. Add 350l of isopropanol into the tube containing the supernatant. Invert about 10 times to mix contents and then keep the tube on ice for 10-15 min.

(Alternatively, you may add 2 volumes of 100% ethanol to the tube and leave it at -20C overnight in order to maximize the plasmid yield. Isopropanol precipipitation is, however, good enough for most applications.)
10. Spin the tube for 10 min. at maximum speed in a microcentrifuge. You should see a transluscent or a whitish-colored precipitate at the bottom. 11. Remove the alcohol by inverting the tube carefully so as to not disturb the pellet. Add 500l of 70% ethanol and vortex tube briefly. Centrifuge for 10 min., and remove ethanol by either gently tipping off the tube or by using a microtip. Spin tube briefly to collect any residual ethanol to the bottom of the tube; remove every drop of liquid using a microtip, without disturbing the pellet. 12. Air-dry the pellet briefly (~5-7 min).

Do not over-dry the pellet, else it may take a long time to dissolve it in the next step.
13. Resuspend the pellet containing plasmid DNA in 50l water or TE buffer. (In

case the pellet does not dissolve leave it in incubator at 37 C for 5 mins. tore at - C). The extracted plasmid can be visualized on a gel.

Optional: RNase A added (a 100X RNase A stock is 20mg/ml). Alternatively, RNase A can be added to the loading dye before running the sample on a gel in order to avoid blazing bands of RNA interfering with the plasmid DNA bands.

Extra facts: STET: Sucrose (It helps to maintain osmolarity so that the cells do not burst open and disrupt the DNA) Tris (Buffer) EDTA ( Helps destabilization of cell membrane, chelation of Mg ions) Triton-X 100 (Neutral detergents that helps break down the cell membrane)

Lysozyme: Helps in breaking the cell wall. Isopropanol/Ethanol: Lowers the dielectric point causing the DNA to precipitate.

ALKALINE LYSIS METHOD Aim: To extract plasmid from bacteria using alkaline lysis method. Theory: Most widely used method of extracting plasmid from cell is by using chemical treatment. The method takes advantage of the fact that genomic DNA gets denatured at pH11.5 -12, but plasmid DNA does not. The cells are gently lysed using alkaline solution, then neutralizing the released contents using a high concentration of potassium acetate ( Refer to 4.1 of Working with DNA). Following extraction phenol chloroform extraction could be performed to purify the DNA. The plasmid, present in supernatant is precipitated using ethanol(100%) (Refer to section 3.5 of Working with DNA). The extracted plamid could be stored in TE buffer which maintains the integrity of the plasmid.

Materials Required: Glassware: Test tube, measuring cylinder, thermometer. Plastic ware: 1.5 ml Microfuge tube, Test tube stand, Float, Micro pipettes (1 ml, 200l, 20l). Reagents: LB broth, Antibiotic (in required concentration), Glucose, 0.5 M EDTA, 1 M Tris-Cl, 10% SDS, 1 N NaOH, 5 M Potassium acetate, Glacial acetic acid, Distilled water, Chloroform:Isoamyl alcohol(24:1), 10 mg/ml RNase A, 100% Ethanol, 70% Ethanol, TE buffer(1 mM EDTA, 10mM Tris-Cl). Misc: Toothpicks, shaking incubator(at 37 C), table top centrifuge machine, water bath(95C), crushed ice. Solution I (GET buffer): Glucose (50 mM),EDTA (10mM), Tris-Cl (25mM) Solution II (freshly prepared): NaOH (0.2 N), SDS (1%) Solution III (10 ml): Potassium acetate(5ml), Glacial acetic acid(1.2ml), Water(2.8ml) Solution IV(10ml):Tris buffered phenol(5ml), Chloroform:Isoamyl alcohol(5ml)

Protocol 1. noculate ml of containing the appropriate antibiotic with a single bacterial colony . incubate with shaking overnight at 37 C.

The bacterial colony used could be transformed E. coli strain DH5 with p R3 , as given in the previous experiment PREPARAT ON AND TRANSFORMATION OF COMPETENT E. COLI USING THE HEAT SHOCK METHOD
2. Fill a 1.5ml microfuge tube with the culture, and spin about 30s to pellet the cells. Discard the supernatant without disturbing the pellet. If culture growth was light, repeat the steop with an additional 1.5ml of culture centrifuged into the same tube. 3. Resuspend the pellet (by vortexing) in 100 l of ice cold solution I. Incubate at room temperature for 5 min. 4. Add 200l of freshly prepared solution II and mix by inverting the tubes 2- 3 times. Incubate on ice for 5 minutes.

( Close the tube and mix the contents by inverting. Make sure that the entire surface of the tube comes in contact with solution II. Do not vortex or increase the time duration, it will damage the DNA. )
5. Add 400 l of ice cold potassium acetate Solution III ad mix gently. Incubate on ice for 5 minutes. (There should no longer be two distinguishable liquid phases. A flocculent white precipitate should form.) 6. Centrifuge at , rpm for min ( C) and transfer the supernatant to new tube. 7. Add 7 l of .5 mg ml RNase A (Diluted from stock of 5 mg ml ). ncubate at 37 C for 30 minutes in water bath. 8. Add 700 l of solution IV. Mix the tube and centrifuge at 10,000rpm minutes ( C). Transfer the aqueous supernatant to a new tube. (Phenol is corrosive in nature, handle with care) 9. Add equal volume of chloroform:isoamyl alcohol ( : ). Mix well and centrifuge at , rpm 5 minutes ( C). Transfer the aqueous supernatant to a new tube. 10. Add two volumes of ice cold 100% ethanol. (In addition, add 1/10th volume of 3M sodium acetate solution for better precipitation of DNA). ncubate at C overnight. 11. Centrifuge at , rpm 5 minutes ( C). Decant the supernatant and invert the tube to allow last drops of supernatant to drain away. 12. Wash the pellet with 500 l of 70% alcohol. Centrifuge at , rpm 5 minutes ( C). Decant the supernatant and air-dry the pellet briefly (~5-7 min). Do not overdry the pellet, else it may take a long time to dissolve in the next step. 13. Resuspend the pellet containing plasmid DNA in 50 l water (or TE). ( n case

the pellet does not dissolve leave it in incubator at 37 C for 5 mins. tore at C). The extracted plasmid can be visualized on a gel.

Extra facts: Solution I is used to lyse the cells and destabilize the cell wall and cell membrane. Solution II increases the pH causing the genomic DNA to denature. SDS acts as detergent. Solution III neutralizes the pH causing the genomic DNA to clump along with cell debris. Phenol chloroform extraction : Proteins in an aqueas phase are folded so that their nonpolar surfaces are hidden, but in phenol they can unfold and still be soluble. Forming an emulsionof the phenol and waterby vigorous mixing will tend to partition the nucleic acids into the aqueous phase and protein into the organic phase. Thus leading to their separation.Excess phenol is removed using chloroform :isoamyl alcohol wash after the purification step. Isopropanol/Ethanol: Lowers the dielectric point causing the DNA to precipitate.

MIDI prep Aim: To extract high quality plasmid using alkaline lysis method on a large scale. Theory : Sometimes for practical purposes we require large quantities of purified DNA. Using a combination of earlier used chemicals in alkaline lysis (Refer to exp no) large amount of DNA can be extracted. Materials Required: Glassware: Test tube, measuring cylinder(10ml, 50ml), thermometer, Pipettes(2ml,5ml). Plastic ware: 50 ml Oakridge tube, Test tube stand, Micro pipettes (1 ml, 200l). Reagents: LB broth, Antibiotic (in required concentration), Glucose, 0.5 M EDTA, 1 M Tris-Cl, 10% SDS, 1 N NaOH, 5 M Potassium acetate, Glacial acetic acid, Distilled water, Chloroform:Isoamyl alcohol(24:1), 10 mg/ml RNase A, 100% Ethanol, 70% Ethanol, TE buffer(1 mM EDTA, 10mM Tris-Cl). Misc: Toothpicks, shaking incubator(at 37 C), table top centrifuge machine, water bath(95C), crushed ice.

Solution I (GET buffer): Glucose (50 mM),EDTA (10mM), Tris-Cl (25mM) Solution II (freshly prepared): NaOH (0.2 N), SDS (1%) Solution III (10 ml): Potassium acetate(5ml), Glacial acetic acid(1.2ml), Water(2.8ml) Solution IV(10ml):Tris buffered phenol(5ml), Chloroform:Isoamyl alcohol(5ml)

Protocol 1. Inoculate 100 ml of LB containing the appropriate antibiotic with a single bacterial colony . ncubate with shaking overnight at 37 C.

The bacterial colony used could be transformed E. coli strain DH5 with p R3 , as given in the previous experiment PREPARAT ON AND TRANSFORMATION OF COMPETENT E. COLI USING THE HEAT HOCK METHOD
2. Spin down the entire culture at 10,000 rpm/5 minutes ( supernatant without disturbing the pellet. C). Discard the

3. Resuspend the pellet (by vortexing) in 2ml of ice cold solution I. 4. Add 500 l of freshly prepared solution of Lysozyme (10 mg/ml). Incubate at room temperature for 20 min. 5. Add 4ml of freshly prepared solution II and mix by inverting the tubes 2- 3 times. Incubate on ice for 5 minutes.

( Close the tube and mix the contents by inverting. Make sure that the entire surface of the tube comes in contact with solution II. Do not vortex or increase the time duration, it will damage the DNA. )
6. Add 3 ml of ice cold potassium acetate Solution III and mix gently. Incubate on ice for 5 minutes. (There should no longer be two distinguishable liquid phases. A flocculent white precipitate should form.) 7. Centrifuge at , rpm for min ( C) and transfer the supernatant to new tube. 8. Add l of mg ml RNase A (Diluted from stock of 5 mg ml ). ncubate at 37 C for 30 minutes in water bath. 9. Add equal volume of solution IV. Mix the tube and centrifuge at , rpm 5 minutes ( C). Transfer the aqueous supernatant to a new tube. 10. Add equal volume of chloroform:isoamyl alcohol ( : ). Mix well and centrifuge at , rpm 5 minutes ( C). Transfer the aqueous supernatant to a new tube.
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11. Add two volumes of ice cold 100% ethanol. (In addition, add 1/10th volume of 3M sodium acetate solution for better precipitation of DNA). ncubate at C overnight. 12. Centrifuge at , rpm 5 minutes ( C). Decant the supernatant and invert the tube to allow last drops of supernatant to drain away. 13. Wash the pellet with 1 ml of 70% alcohol. Centrifuge at , rpm 5 minutes ( C). Decant the supernatant and air-dry the pellet briefly (~5-7 min). Do not overdry the pellet, else it may take a long time to dissolve in the next step. 14. Resuspend the pellet containing plasmid DNA in 250 l water (or TE). ( n

case the pellet does not dissolve leave it in incubator at 37 C for 5 mins. tore at - C). The extracted plasmid can be visualized on a gel.