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-   -   Plasmid DNA Isolation from Bacteria (http://www.molecularstation.com/forum/molecular-biology-articles-protocols/218-plasmid-dna-isolation-bacteria.html)

domba 07-31-2006 11:04 PM

Plasmid DNA Isolation from Bacteria
 
Materials:

1. TENS solution:
* 10 mM Tris (pH to 7.5)
* 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
* 0.1 N sodium hydroxide
* 0.5 % sodium dodecyl sulfate
2. 3 M Sodium acetate, pH 5.2
3. Pre-chilled (at -20 degrees C) 100 % ethanol
4. 70 % Ethanol
5. Distilled water
6. Overnight bacterial culture

Supplies:

1. Micropipetter and tips
2. Vortex mixer
3. Microcentrifuge and tubes

Procedures:

1. Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.)
2. Decant supernatant, leaving 50-100 ul in the tube.Teacher's note: In order to resuspend cells completely, some supernatant should be left, but too much will dilute out the TENS solution.
3. Vortex to resuspend the bacteria pellet completely.
4. Add 300 ul of TENS solution.
5. Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.)Teacher's note: If more than 10 minutes are needed before moving on to the next step, the tube should be set on ice/ice water bath to prevent degradation of bacterial chromosomal DNA which can then coprecipitate with plasmid DNA.
6. Add 150 ul of the sodium acetate.
7. Vortex for 5 seconds to mix.
8. Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.)
9. Transfer supernatant to a fresh tube.
10. Add 0.9 ml of pre-chilled 100 % ethanol.
11. Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)
12. Discard supernatant and add 1 ml of 70 % ethanol.
13. Discard the ethanol and add another 1 ml of 70 % ethanol.
14. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
15. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.Teacher's note: DNA should be stored at -20 degrees C, but repeated freeze-thaw should be avoided. So if it is needed frequently or in a couple of days, store it at 4 degrees C.

Results:

1. Plasmid DNA is now ready for estimation of DNA concentration followed by restriction digest.
2. Typical yield is 2-3 ug.

shreenathnayak 08-04-2008 05:14 AM

Re: Plasmid DNA Isolation from Bacteria
 
please send a very good protocol for bacterial plasmid aroun 9 to 10 kb isolation

migandi 03-14-2011 03:08 PM

Re: Plasmid DNA Isolation from Bacteria
 
Quote:

Originally Posted by domba (Post 498)
Materials:

1. TENS solution:
* 10 mM Tris (pH to 7.5)
* 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
* 0.1 N sodium hydroxide
* 0.5 % sodium dodecyl sulfate
2. 3 M Sodium acetate, pH 5.2
3. Pre-chilled (at -20 degrees C) 100 % ethanol
4. 70 % Ethanol
5. Distilled water
6. Overnight bacterial culture

Supplies:

1. Micropipetter and tips
2. Vortex mixer
3. Microcentrifuge and tubes

Procedures:

1. Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.)
2. Decant supernatant, leaving 50-100 ul in the tube.Teacher's note: In order to resuspend cells completely, some supernatant should be left, but too much will dilute out the TENS solution.
3. Vortex to resuspend the bacteria pellet completely.
4. Add 300 ul of TENS solution.
5. Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.)Teacher's note: If more than 10 minutes are needed before moving on to the next step, the tube should be set on ice/ice water bath to prevent degradation of bacterial chromosomal DNA which can then coprecipitate with plasmid DNA.
6. Add 150 ul of the sodium acetate.
7. Vortex for 5 seconds to mix.
8. Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.)
9. Transfer supernatant to a fresh tube.
10. Add 0.9 ml of pre-chilled 100 % ethanol.
11. Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)
12. Discard supernatant and add 1 ml of 70 % ethanol.
13. Discard the ethanol and add another 1 ml of 70 % ethanol.
14. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
15. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.Teacher's note: DNA should be stored at -20 degrees C, but repeated freeze-thaw should be avoided. So if it is needed frequently or in a couple of days, store it at 4 degrees C.

Results:

1. Plasmid DNA is now ready for estimation of DNA concentration followed by restriction digest.
2. Typical yield is 2-3 ug.

Could you tell me, what the reference of this protocol is? or Are you the author? Thanks.

Sincerely.


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