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DNA Plasmid Isolation

The document describes the alkaline lysis method for plasmid DNA isolation from bacterial cells. It involves three alkaline lysis solutions - Solution I contains glucose, Tris, and EDTA; Solution II contains sodium hydroxide and SDS; Solution III contains potassium acetate. Bacterial cells are lysed with Solutions I and II, then neutralized with Solution III. The plasmid DNA is purified by ethanol precipitation and dissolved in TE buffer for analysis by agarose gel electrophoresis.

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Mahtab Ahmad
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239 views3 pages

DNA Plasmid Isolation

The document describes the alkaline lysis method for plasmid DNA isolation from bacterial cells. It involves three alkaline lysis solutions - Solution I contains glucose, Tris, and EDTA; Solution II contains sodium hydroxide and SDS; Solution III contains potassium acetate. Bacterial cells are lysed with Solutions I and II, then neutralized with Solution III. The plasmid DNA is purified by ethanol precipitation and dissolved in TE buffer for analysis by agarose gel electrophoresis.

Uploaded by

Mahtab Ahmad
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA Plasmid Isolation Using Alkaline Lysis Method

Buffers and Solutions


Alkaline lysis solution I: 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0), de-
ion water
Alkaline lysis solution II : 0.2 N NaOH, 1% (w/v) SDS, de-ion water
Alkaline lysis solution III : 5 M potassium acetate, glacial acetic acid, de-ion water
Ethanol 70% (v/v)
Isopropanol
TE-RNAase pH 8.0

Method
1. Pour overnight grown culture to 1.5 mL labeled falcon tube.
2. Centrifugate at 14.000 rpm for 1 min.
3. Remove the supernatant from the tube.
4. Repeat step 1-3, until leaves bacterial pellet as dry as possible.
5. Add 150 L resuspension buffer, resuspend the bacterial pellet properly by
vortexing.
6. Add 200 L lysis solution to bacterial suspension (freshly made), close the tube
tightly and mix contents thoroughly by inverting the tube 4-6 times until the solution
becomes viscious.
7. Add 300 L neutralization solution and mix contents thoroughly by inverting the
tube 4-6 times.
8. Centrigufe at 14.000 rpm for 5 min.
9. Take the supernatant and transfer to a new 1.5 mL falcon max 300 L.
10. Add equal volume of isopropanol in the supernatant (300 L) and mix it by inverting
the tube couple of times.
11. Incubate in -80C for 30 min.
12. Centrifuge at 14.000 for 5 min.
13. Remove the supernatant and add 600 L EtOH 70%.
14. Centrifuge at 14.000 for 5 min.
15. Remove the supernatant and dry the pellet for 10-30 min.
16. Dissolve the pellet in 20-50 L TE-RNAase pH 8.0. Confirm the plasmid with 5 L DNA
solvent by Agarose Electroforessis.

Recipes
Alkaline lysis solution I
1. 1 M glucose stock solution (50 mL)
a. Dissolve 9 gram of glucose in 50 mL sterilized de-ion water.
b. Filter sterilize using membrane millipore (0.20 M).
c. Glucose solution is ready to use or store at 4C cabinet for preservation.
2. 1 M Tris-Cl stock solution (50 mL)
a. Dissolve 6.057 gram of Tris base in 50 mL sterilized de-ion water.
b. Adjust the pH to the desired value by adding concentrated HCl.
3. 0.5 M EDTA stock solution (100 mL)
a. Dissolve 14.612 gram of EDTA in 100 mL sterilized de-ion water.
b. Adjust the pH to 8.0 with NaOH.
Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15
minutes at 15 psi and store at 4C.
Alkaline lysis solution I Volume
1 M Glucose 5 mL
1 M Tris-Cl 2.5 mL
0.5 M EDTA 1 mL
De-ion water 90.5 mL
Total volume 100 mL

Alkaline lysis solution II


1. 10 N NaOH stock solution (50 mL)
Dissolve 20 gram of NaOH in 50 mL sterilized de-ion water.
2. 1% (w/v) SDS stock solution (30 mL)
Dissolve 0.3 gram of SDS in 30 mL sterilized de-ion water.
Prepare Solution II fresh and use at room temperature.
Alkaline lysis solution II Volume
0.2 N NaOH 200 L
1% SDS 1 mL
De-ion water 8.8 mL
Total volume 10 mL

Alkaline lysis solution III


1. 5 M potassium acetate stock solution (100 mL)
Dissolve 49.071 gram of potassium acetate in 100 mL sterilized de-ion water.
Store the solution at 4C and transfer it to an ice bucket just before use.
Alkaline lysis solution III Volume
5 M Potassium acetate 60 mL
Glacial acetic acid 11.5 mL
De-ion water 28.5 mL
Total volume 100 mL
Reference
Sambrook, J., Fritsch, E.F., dan Maniatis, T. 1989. Molecular Cloning A Laboratory Manual
2nd edition. New York : Cold Spring Harbor Laboratory Press.

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