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Plasmid Extraction Protocol (1)

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12 views3 pages

Plasmid Extraction Protocol (1)

Uploaded by

ayeshashaban065
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Plasmid Extraction Protocol

Materials and Reagents:


1. E. coli culture containing the plasmid of interest.
2. Sterile LB or other appropriate growth medium.
3. Sterile 1.5 ml microcentrifuge tubes.
4. Sterile pipettes and tips.
5. Bacterial centrifuge with appropriate rotor.
6. Ice or a refrigerated centrifuge.
7. Tris-EDTA (GTE) buffer (Glucose, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
8. Alkaline lysis solution (e.g., 0.2 N NaOH, 1% SDS).
9. Neutralization solution (e.g., 3 M potassium acetate, pH 5.5).
10. Isopropanol.
11. Ethanol (70% and 100%).
12. Sterile distilled water.
13. Microcentrifuge.
Procedure:
1. Start by inoculating a single colony of E. coli containing the plasmid of interest into 3-5
ml of LB medium supplemented with the appropriate antibiotic for plasmid selection.
Incubate the culture overnight at 37°C with shaking.
2. Transfer 1-2 ml of the overnight culture into a sterile 1.5 ml microcentrifuge tube.
3. Centrifuge the tube at maximum speed (usually 12,000-14,000 x g) for 1-2 minutes to
pellet the bacterial cells.
4. Carefully decant and discard the supernatant.
5. Resuspend the bacterial pellet in 100-200 µl of GTE buffer.
6. Add 200 µl of alkaline lysis solution and mix gently by inverting the tube several times.
7. Incubate the mixture on ice for 5-10 minutes. Do not exceed 10 minutes, as this can
damage the plasmid DNA.
8. Add 150 µl of neutralization solution and mix immediately by inverting the tube several
times. A white precipitate should form.
9. Centrifuge the tube at maximum speed for 5 minutes to pellet cell debris and precipitated
proteins.
10. Transfer the supernatant (containing plasmid DNA) to a fresh microcentrifuge tube.
11. Add an equal volume of isopropanol (e.g., 200 µl) to the supernatant and mix gently.
12. Centrifuge at maximum speed for 5-10 minutes to pellet the plasmid DNA.
13. Carefully remove the isopropanol and wash the DNA pellet with 70% ethanol.
14. Centrifuge briefly and remove the ethanol.
15. Allow the DNA pellet to air-dry for a few minutes to remove any remaining ethanol.
16. Resuspend the DNA pellet in an appropriate volume of sterile distilled water or TE
buffer.
Alkaline Lysis Solution (e.g., 0.2 N NaOH, 1% SDS):

This solution disrupts the bacterial cell membrane, denatures proteins, and solubilizes genomic
DNA, leaving plasmid DNA intact.
Ingredients:
0.2 N NaOH: To make 0.2 N NaOH, dissolve 8 g of NaOH (sodium hydroxide) in 100 ml of
distilled water. Always add NaOH to water slowly and with caution, as it generates heat. Wear
appropriate personal protective equipment (PPE), such as gloves and safety goggles, when
handling NaOH.
10% SDS (Sodium Dodecyl Sulfate): For 1% SDS, dilute 10% SDS 1:10 with distilled water.
So, for example, mix 10 ml of 10% SDS with 90 ml of distilled water to make 100 ml of 1%
SDS.
Procedure:
Mix 1 volume of 0.2 N NaOH with 1 volume of 1% SDS. For example, if you need 2 ml of
alkaline lysis solution, mix 1 ml of 0.2 N NaOH with 1 ml of 1% SDS.
Mix the solutions thoroughly by gentle inversion.
Neutralization Solution (e.g., 3 M potassium acetate, pH 5.5):
The neutralization solution is used to precipitate proteins and genomic DNA, allowing plasmid
DNA to remain in the supernatant after centrifugation.
Ingredients:
3 M Potassium Acetate (KCH3CO2): To make 3 M potassium acetate, dissolve 186.2 g of
potassium acetate in 100 ml of distilled water. Adjust the pH to 5.5 using glacial acetic acid
(CH3COOH). You may need to titrate the pH carefully to achieve the desired value.
Procedure:
Prepare 3 M potassium acetate solution with a pH of 5.5 as described above.
Store the neutralization solution at room temperature.

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