DNA ISOLATION FOR PLASMID

DNA ISOLATION FOR PLASMID ISOLATION

Reagents and solutions

1. Alkaline Solution I
50 mM glucose
25 mM Tris HCl (pH 8.0)
10 mM EDTA (pH 8.0)

2. 10 mg/ml lysozyme

3. Alkaline Solution II
0.2 N NaOH (2.0 ml of 10 N NaOH in 100ml double distilled water)
1.0% SDS

4. Alkaline Solution III
5 M Potassium acetate
Glacial acetic acid
Autoclaved water

5. TE buffer
10 mM Tris HCl (pH 8.0)
1.0 mM EDTA (pH 8.0)

6. Ring stand with clamp

7. 26 gauge needle

8. 1 cc syringe

9. Dialysis buffer
1 M Tris HCl (pH 7.5)
0.5 M EDTA (pH 8.0)
Autoclaved distilled water (1L)

10. Ampilcillin 100 µg/ml

11. Ice cold Isopropyl alcohol

12. Cesium chloride as per Plasmid DNA sample

13. Dialysis tubings (Nylon)

14. 5 M NaCl
29.22 g NaCl in 75 ml water and bring the volume to 100 ml.

15. Isoamyl alcohol

16. Luria Broth media
1 gm Tryptone
0.5 gm Yeast extract
1 gm Sodium chloride
Deionised water 75 ml
Shake all the solutes until dissolved. Adjust the pH to 7.0 with 5 N NaOH. Bring the volume of the solution to 100 ml with deionised water and sterilize it by autoclaving it for 20 minutes at 15 psi.

Procedure

Isolation of Plasmid DNA

1. Pick up a colony of bacteria and inoculate it in a conical flask containing 100 ml autoclaved Luria broth media supplemented with antibiotic (Ampicillin 100 µg/ml) and incubate overnight in a 37°C shaking water bath at 250 rpm.
2. Pour the culture in a 2.0 ml centrifuge tube and centrifuge at 5000 rpm for 20 minutes.
3. Discard the supernatant and wash the pellet in 1.0 ml double distilled water. Centrifuge the solution at 1000 rpm for 5 minutes.
4. Discard the supernatant and resuspend the pellet in 250µl cold alkaline solution I (stored at 4°C). Mix properly so that the pellet dissolves.
5. Add 20 mg of lysozyme in the above solution and mix well. Allow it to incubate on ice for 15 minutes.
6. Add 500 µl of alkaline solution II and mix properly. Mix the solution by flicking.
7. Now add 250 µl of alkaline solution III and incubate it on ice for 15 minutes.
8. Centrifuge the sample at 5000 rpm for 10 minutes and filter the supernatant into an autoclaved 2.0 ml centrifuge tube.
9. Fill the tube with the same amount of ice-cold isopropyl alcohol (stored at-20°C) and incubate in cold (-20°C) for 30 minutes.
10. Centrifuge the sample at 5000 rpm for 15 minutes. Discard the supernatant and air dry the pellet.
11. Resuspend the pellet in 500 µl TE buffer.

Purification of Plasmid DNA using Cesium Chloride

1. When the pellet is suspended completely, measure the mass of plasmid DNA by using a balance. For every gram of plasmid DNA solution, add 1.0 gm of solid cesium chloride. Warm the solution at 30°C to dissolve the CsCl salt.
2. Add 5 µl of 10mg/ml ethidium bromide to the above plasmid DNA solution.
3. Centrifuge the sample at 50,000 rpm for 10-15 hours at 20°C in an ultracentrifuge.
4. After the first run is complete remove the tube carefully and place it in the clamp and insert a needle just below the band. Remove the cap on the tube and pull out the band with the help of needle and syringe.
5. Place the sample in another ultra centrifuge tube and fill the tube with a solution of cesium chloride and TE. Spin at 50,000 rpm for 5 hours.
6. When the run is complete follow the above instructions for removing the bands.
7. Place the bands in a centrifuge tube and add equal volumes of TE and mix well. Now add isoamyl alcohol twice the volume of sample in the tube. Shake well and allow the phases to separate.
8. Pipet off the top pink layer and repeat the extraction with isoamyl alcohol until the top layer is clear and transparent.


Dialysis of Plasmid DNA

1. Prepare pieces of dialysis tubing by rinsing very well in autoclaved distilled water. Tie one end of the tube and carefully suspend he extracted plasmid DNA band into the tubing. Tie the other end too and place the sample in dialysis buffer.
2. Dialyze for at least 6 hours at 4°C using a magnetic stirrer. Repeat the process in new buffer solution.
3. Once the dialysis is complete, remove the liquid from the tubing and transfer it into another tube.
4. Add 1/10 volume of 5 M NaCl and twice the volume of pure ethanol.
5. Centrifuge the samples for 20 minutes at 5000 rpm at 4°C.
6. Discard the supernatant, leaving the pellet.
7. Rinse with 95% Ethanol and resuspend the pellet in 100 μl of TE buffer (pH 8.0) or autoclaved distilled water.