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United States Department of Agriculture

Agricultural Research Service

DNA Extraction by CsCl Ethidium Bromide SOPs
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STANDARD OPERATING PROCEDURE FOR DNA EXTRACTION BY CsCl ETHIDIUM BROMIDE

Reviewed - 4/8/98

Contact [deleted]

Materials Needed:

1. CsCl (Irritant)

2. TES buffer (500 ml) - 10 X TE buffer pH 8.0 - 50 ml;

NaCl - 4.12 g;

Adjust by distill. H2O till 500 ml

3. 1 x TE buffer

4. Proteinase K (solution) -20 mg/ml. Store in the freezer at -20OC in little volumes

5. 10% SDS (Irritant, sensitizer)

6. Phenol/SEVAG 1:1 - prepare the day before using (Phenol is highly toxic, causes burns)

7. SEVAG (Chloroform/Isoamyl alcohol - 24:1) (Chloroform is a highly toxic mutagen and is a carcinogen. Isoamyl alcohol is harmful, risk of serious eye damage and is combustible.)

8. 95% EtOH (Irritant, flammable)

9. 70% EtOH

10. CTAB/NaCl solution (10% CTAB in 0.7 M NaCl) (CTAB is toxic, corrosive and harmful.)

Dissolve 4.1 g NaCl in 80 ml water and slowly add 10 g CTAB (Hexadecyltrimethyl ammonium bromide) while heating and stirring. If necessary, heat to 65OC to dissolve.

Adjust final volume to 100 ml.

11. Nutrient Broth

12. CsCl - saturated Isopropanol solution: (Isopropanol is an irritant and is flammable.)

Take 10 - 15 g CsCl, dissolve in 5 - 10 ml TE buffer. You have to see a few crystals of CsCl on the bottle of the tube. Add 20 ml of Isopropanol. Keep in room temperature.

13. Ethidium bromide - 10 mg/ml (Powerful mutagen)


Personal Protective Equipment

1. Nitrile gloves for Ethidium bromide use.

2. Vinyl or latex gloves changed frequently for Phenol/Chloroform and Isoamyl alcohol (Not resistant to Chloroform.)

3. Lab coat

4. UV eye protection

5. Splash goggles

6. Read all pertinent MSDS sheets


Safety, Health and Chemical Hygiene Requirements for Conducting this Protocol:

1. Ethidium bromide is a powerful mutagen and a moderately toxic irritant and sensitizer. Gloves must be worn when working with the CsCL gradients containing highly concentrated solutions of EtBr. After each handling of solutions containing EtBr, contaminated gloves must be discarded into a clean plastic bag and disposed of in the medical waste container, to prevent the spreading of EtBr to adjacent areas of the work bench.

2. The following must be collected and disposed of as Hazardous wastes; profile #USDA 0375 - Chloroform/Isoamyl alcohol/Phenol, profile # USDA 0533 - Ethanol/Ethidium Bromide/Isopropanol/etc., profile # USDA 0037 - Cesium Chloride/EtBr gradient, and profile # USDA 0435 - EtBr/Cesium Chloride solid waste.

3. Ultraviolet (UV) radiation is dangerous to exposed skin, and especially the eyes. Always wear a full UV protective face shield.

4. Dispose of absorbent bench pad in EtBr solid waste if contaminated. Check benchtop with hand held UV light, and clean up any EtBr spills with soap and water.

5. Use Chloroform, Phenol, and Isoamyl alcohol in a fume hood. Wear proper gloves, and eye protection.


Preparation and Lysis of Cells

1. Grow bt from single colony in the tube (5 ml Nutrient Broth) on the shaker in 27OC incubator overnight at 150 rpm.

2. Transfer 1 ml of the liquid culture to the Fernback flask containing 1000 ml NB media.

3. Set Fernback flask on shaker on 120 rpm and 27OC overnight.

4. Take a loop of this liquid from Fernback flask and streak a petri plate containing agar YDC. Set streaked petri plate in 27oC incubator overnight to see if there is any contamination in your sample.

5. Divide your liquid culture (1000 ml) in the 4 sterile containers and get the pellets from them.

6. Wash each pellet subsequently by 100 ml 1 x TES buffer.

7. Spin your pellet in the last tube at 5,000 rpm for 5 min. Discard supernatant.

8. Resuspend your pellet in 100 ml of TE buffer and take from tube 2 - 50 ml aliquots.

9. Keep the liquid in the freezer at -20oC.


Precipitation and Purification of DNA

1. To 5 tubes add 10 ml of pellet in TE buffer. Add 0.5 ml of 10% SDS and 50 ul of 20 mg/ml Proteinase K. Mix thoroughly and incubate 1 hour at 37oC.

2. Add 1.8 ml of 5 M NaCl and mix thoroughly.

3. Add 1.5 ml CTAB/NaCl solution. Mix thoroughly and incubate 20 min at 65oC.

4. Extract with equal volume of SEVAG. Centrifuge at 6,000 x g (JA-20 rotor at 7,000), room temperature for 10 min, to separate phases.. Use a hood when working with SEVAG and Phenol and use gloves and a lab coat and splash goggles.

5. Transfer aqueous supernatant to a fresh tube using a wide-bored pipet.

The supernatant will probably be very viscous if the yield is high. An additional Phenol/SEVAG extraction, is optional but should not be necessary if the material is to be purified on a CsCl gradient.

6. Collect SEVAG for disposal using waste profile # USDA 0375.

(This item is optional)

15. Add an equal volume of Phenol/SEVAG. Extract thoroughly. Spin 10 min at 8,000 rpm. Collect Phenol/SEVAG and dispose of using waste profile # USDA 0375.

16. Add 0.6 vol isopropanol and mix until a stringy white DNA pellet precipitates out of solution and condenses into a tight mass. Remaining solution dispose of using waste profile # USDA 0533.

17. Transfer the precipitate to 1 ml of 70% EtOH in a fresh tube, by hooking it on the end of Pasteur pipet that has been bent and sealed in a Bunsen flame.

18. Spin the pellet in 70% EtOH 5 min at 10,000 x g (JA-20 rotor at 9900 rpm). Remove supernatant and redissolve pellet in 4 ml TE buffer. This may take several hours (2 - 3 hours at 60oC) to overnight (at 4oC). Collect 70% Ethanol using waste profile # USDA 0533.

19. Add 4.3 g CsCl per 4 ml TE buffer. Dissolve. Add 200 ul of 10 mg/ml Ethidium bromide. Transfer to 4/ml sealable centrifuge tubes. Adjust volume and balance tubes. Seal tubes. Centrifuge in Ti80 rotor at 60,000 rpm for 48 hrs. at 15oC. Use gloves and a lab coat when working with Ethidium bromide. Cover work area with plastic film (like Saran Wrap) and absorbing diapers to contain any Ethidium bromide that might spill. Monitor with hand held UV light. In all procedures using Ethidium bromide discard this material in a separate container as Ethidium bromide solid waste profile # USDA 0435. Change gloves frequently.

20. Visualize gradient under longwave UV lamp wearing UV eye protection. A single band should be visible. Remove band using a 18 g needle and 3-ml plastic syringe. Use caution to avoid finger or hand needle puncture. Do not replace cap and dispose of needle in sharps container. If the DNA is intact high-molecular weight chromosomal DNA it will appear very viscous as the band is withdrawn from the gradient; hence, it is important to use a wide-bore needle to avoid mechanical shearing of DNA. If the band appears right at the top of the gradient, then the gradient is too dense. Reduce the amount of CsCl added in step 20. Collect gradient waste for disposal using waste profile # USDA 0037. Dispose of MT tube in EtBr solid waste profile # USDA 0435.

21. Remove the ethidium bromide by sequential extractions with CsCl-saturated in TE/Isopropanol (1:1). Collect waste and dispose using waste profile # USDA 0533.

Extract until the pink color disappears.

22. Dialyze overnight against 2 liters TE buffer or HOH to remove CsCl.

23. Measure the DNA concentration on a spectrophotometer.

 

Standard Operating Procedure for Purification of Bacterial Plasmid DNA CsCl Gradients, using Ethidium Bromide to Visualize Nucleic Acid Band

Reviewed - 4/8/98

[deleted]

Materials Needed Personal Protective Equipment

STET buffer Lab coat

Lysozyme Six pairs of Nitrile gloves

Polyethene Glygol 8000 UV protective face shield

Cesium Chloride ultrapure (Irritant) Eye protection

Mineral oil Read all pertinent MSDS

Ethidium Bromide (Powerful mutagen) Safety sheets

Absorbent Bench Pad

Isoamyl alcohol (Harmful, risk of serious eye damage)

Ammonium Acetate (Irritant)

ETOH (Irritant, flammable)


Safety, Health and Chemical Hygiene Requirements for Conducting this Protocol:

1. Ethidium Bromide is a powerful mutagen and a moderately toxic irritant and sensitizer. Gloves must be worn when working with the CsCl gradients containing highly concentrated solutions of EtBr. After each handling of solutions containing EtBr, contaminated gloves must be discarded into a clean plastic bag and disposed of in the medical waste container, to prevent the spreading of EtBr to adjacent areas of the work bench.

2. All wastes containing EtBr must be collected and disposed of as Hazardous wastes, using the following waste profile #0037 Cesium chloride / EtBr gradient waste, #0032 Isoamyl alcohol with EtBr, and #0435 EtBr solid waste.

3. Ultraviolet (UV) radiation is dangerous to exposed skin, and especially the eyes. Always wear a full UV protective face shield.

4. Dispose of absorbent bench pad in EtBr solid waste if contaminated. Check benchtop with hand held UV light, and clean up any EtBr spills with soap and water.

5. Use Isoamyl alcohol in a fume hood, wear nitrile gloves and eye protection.

Day 1 - AM

1. Inoculate 2.5 ml of LB (with antibiotic) with a single colony from a freshly streaked LB (with antibiotic plate).

2. Grow for 6-8 hrs, add 2 ml to 200 ml of LB (with antibiotic) in a liter flask, and grow overnight (200 RPM) at 37oC.

Day 2 - AM

3. Pellet the cells by centrifuging at 5 K RPM in the GSA rotor (4,000 x g) for 15 minutes at 4oC. Pour off the media. Drain the residual media by inverting the centrifuge bottles on paper towels for 3 minutes.

4. Gently suspend the pellet in 25 ml of STET buffer (room temperature) using a 25 ml pipet (minimizing air bubbles). Transfer to a 125 ml flask and add 2.5 ml of a freshly prepared 10 mg/ml solution of lysozyme (Sigma No. L-6876) prepared in STET buffer. Swirl and incubate for 5 minutes at room temperature. While wearing a protective glove, immerse the bottom of the flask in a boiling water bath for 45 seconds, swirling continuously. Transfer immediately to an ice water bath and swirl for 1 minutes. Incubate on ice for an additional 2 minutes.

5. Slowly pour the viscous mixture into a 50 ml centrifuge tube. Pellet the bacterial debris by centrifuging at 11.5 K RPM in the SS-34 rotor (16,000 x g) at 4oC for 20 minutes Transfer the supernatant to a 50 ml centrifuge tube.

6. Estimate the volume and, per 10 ml, add 1 ml 5.0 M NaCl and 1 g PEG-8000 (Sigma No. P-2139) Mix slowly by hand inversion until the PEG is completely dissolved (15-20 mins). Incubate on ice (at least 2 hours)

Day 2 - PM

7. Pellet by centrifuging in the GPR at 3.8 K RPM (3,000 x g) at 4oC for 10 mins. Drain off the supernatant and gently suspend the pellet in 2.5 ml 1 X TE (pH 7.5). Add 25 ul 10 mg/ml Rnase A and incubate at 37oC for 30 minutes.

8. Adjust the volume exactly to 4.0 ml with 1X TE. Add 4.1 g CsCl and gently swirl until the CsCl dissolves. Add 171 ul 10 mg/ml ethidium bromide (EtBr) and mix gently. Remember to use protective gloves for the remainder of the protocol through Day 3. Use a designated area for EtBr use.

9. Transfer to a polyallomer 16 x 67 mm Quick-Seal tube (Beckman No. 344622). Overlay with mineral oil and heat seal.

10. Centrifuge in the 70.1 Ti rotor at 45 K RPM at 20o C for 13-15 hours.

11. If the yield is good, there should be two separated bands of DNA visible (without the aid of a UV lamp). Puncture the top of the tube with an 18 gauge needle and then puncture the bottom of the tube with the needle. Use caution to avoid finger or hand needle puncture. Do not replace cap and dispose of needle in sharps container. Collect the lower band directly into a 15 ml polyethylene centrifuge tube. If you collect more than 0.5 ml, subdivide into two equal aliquots (in separate 15 ml tubes). Do not collect the upper band which is a mixture of linear plasmid, nicked plasmid, and chromosomal DNA. Remember to change gloves frequently and carefully collect EtBr waste and tubes for disposal using hazardous waste profile #0037.

12. Add sterile double-distilled water to bring the volume in the tube(s) to 6 ml. Extract the EtBr from the DNA using an equal volume of isoamyl alcohol (3 mins). Centrifuge in the GPR (3.8 K RPM, 5 mins) to separate the phases and discard the upper phase (the aqueous phase will be on the bottom!). Add more isoamyl alcohol and repeat the extraction. Three total extractions are usually sufficient. After the final extraction, the aqueous phase should be colorless. Carefully collect and dispose of EtBr saturated Isoamyl alcohol using hazardous waste profile #0032.

13. There should be about 5-6 ml of DNA solution in the tube (if not, add water to make a final volume of 5 ml). Add 9-10 ml of 95% ethanol to each tube and mix thoroughly. Store at -20oC (overnight).

Day 4

14. Recover the DNA by centrifuging at 3.8 K RPM in the GPR centrifuge at 4oC for 30 minutes. Pour off the ethanol and invert the tube on paper towels for a few minutes.

15. Add 300 ul sterile double-distilled water to each pellet and incubate on ice for 15 mins. Gently finger-tap the tube to get the DNA into solution. Transfer to a sterile 1.5 ml microfuge tube. Add 150 ul 7.5 M ammonium acetate (pH 7.5) followed by 900 ul ethanol. Mix and store at -20o C (overnight).

Day 5

16. Recover the DNA by microcentrifugation (12,000 g) at 4oC for 15 minutes. Rinse the pellet once with 70% ethanol (-20oC). Remove the ethanol with pasteur pipet. Microfuge for 10 seconds (room temperature) and remove the residual ethanol with a micropipettor. Add 100-300 ul sterile double-distilled water. Incubate on ice for 15 minutes. Gently finger-tap the tube to get the DNA into solution. Conduct spectrophotometry to estimate the DNA concentration. Store at -20oC (or 4oC).


Last Modified: 4/29/2005
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