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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Microbiology > Microbial genetics > DNA
Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
2/5/2011, 49228 views, 30 Q&A

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits.

Materials and Reagents

  1. RNAase (Life Technologies, Invitrogen™)
  2. Isopropanol (EM Science)
  3. Ethanol (VWR Chemical)
  4. Tryptone
  5. Yeast extract
  6. NaCl
  7. Glucose
  8. EDTA
  9. 0.2 N NaOH
  10. SDS
  11. KOAc
  12. Potassium acetate
  13. Glacial acetate
  14. Tris-HCl (pH 8.0)
  15. Luria-Bertani broth (LB) medium: Bacto-tryptone (BD Biosciences), yeast extract (BD Biosciences) (see Recipes)
  16. Resuspension solution (P1 buffer) (see Recipes)
  17. Lysis solution (P2 buffer) (see Recipes)
  18. Neutralizing solution (P3 buffer) (see Recipes)
  19. TE (see Recipes)

Equipment

  1. Table-top centrifuges
  2. 1.5-ml eppendorf tube
  3. 37 °C heat blocker

Procedure

  1. Grow bacterial (E. coli) culture in LB medium with appropriate antibiotics at 37 °C overnight (O/N) with shaking. For >10 copies plasmid, 3 ml cell culture is usually enough.
  2. Transfer O/N culture to a 1.5-ml eppendorf tube, and spin down cell culture (twice) at high speed for 1 min at table-top centrifuge.
  3. Discard the supernatant. To remove the liquid completely by upside down tube onto a piece of paper towel for a few sec.
  4. Add 100 μl of resuspension solution (P1 buffer) into each tube, and vortex to completely resuspend cell pellet
  5. Add 100 μl of lysis solution (P2 buffer) and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial
    lysis has taken place.
  6. Add 150 μl of neutralizing solution (P3 buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
  7. Centrifuge the tubes at high speed for 10 min.
  8. Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5-ml eppendorf tube with a 1ml pipette.
  9. Add 2.5-3 volume of 200-proof cold ethanol (stores at -20 °C) to each tube and mix by inverting the tubes a few times.
  10. Spin down plasmid DNA precipitate (transparency pellet) at high speed for 10 min.
  11. Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns white when dry.
  12. Resuspend the DNA pellet with 50 μl TE. Completely dissolve the pellet by pipetting solution several times.
    Note: Large amounts of RNA is present in the DNA sample. Therefore, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to the resuspension solution with a final concentration of 1 mg ml-1.

Recipes

  1. LB medium
    1% Tryptone
    0.5% yeast extract
    200 mM NaCl
  2. Resuspension solution (P1 buffer)
    50 mM glucose
    10 mM EDTA
    25 mM Tris (pH 8.0)
    Store at 40 °C.
  3. Lysis solution (P2 buffer)
    0.2 N NaOH
    1% SDS
    Store at room temperature.
  4. Neutralizing solution (P3 buffer)
    3 M KOAc (pH 6.0)
    For 100 ml solution, 60 ml 5 M potassium acetate (49.07 g potassium acetate in 100 ml H2O)
    11.5 ml glacial acetate and 28.5 ml H2O, store at room temperature.
  5. TE
    1 mM EDTA
    10 mM Tris-HCl (pH 8.0)
    Note: P1, P2, P3 buffers from the QIAGEN DNA extraction kit also work well.

References

  1. Birnboim, H. C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6): 1513-1523.
  2. Birnboim, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol 100: 243-255.


How to cite this protocol: (2011). Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method. Bio-protocol Bio101: e30. http://www.bio-protocol.org/e30



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Anonymous

8/31/2011 2:49:53 PM  

why a cold temperature is maintained through out the process?

Fanglian

8/31/2011 3:48:38 PM  

All steps except of steps 9 and 10 are carried out at RT.

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2/2/2012 11:42:42 PM  

how i isolate a plasmid for constructing a metagenomic library.is it sam procedure or different.can u tell me exact protocol for construction of library from soil ecosystem

Fanglian

2/3/2012 1:54:27 PM  

Depends on which bacteria you want to isolate plasmid from. It should be very similar, expect of cell lysis (step4), which may require additional treatment to open the cell. For instance, to isolate plasmid from Agrobacterium, it usually adds lysozyme at lysis step. Since it is fair quick procedure once you have solutions ready (you can use solutions from commercial mini prep kits), I’d suggest to try it using this protocol and see if it works. Also, if your plasmid is low copy (<10copies), you may need lager cell culture (like 50 ml) to start with, and scale up all solutions used in the protocol (for example, 50 ml cell culture, use 1ml solution I ).

I think my experience with library construction was not solid enough to provide a protocol here. But, we will invite an author who has successful experience with this experiment to contribute the protocol to our database soon, will keep you updated.

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3/8/2012 7:08:30 PM  

i want plasmid dna isolation from psuedomonas.please help me

Fanglian

3/10/2012 4:39:48 AM  

I do not have experience of plasmid DNA isolation from Psuedomonas. But I asked one of my friends who is an expert in bacterial pathogenesis in plants, here is his answer:

"I used to isolate plasmid from Pseudomonas strains with normal alkaline lysis method with phenol/chloroform extraction for E.coli. It worked well for small plasmid, but if the size of the plasmid is too big, I used classical Kado method (attached file). When I really need a lot and pure plasmid DNA, I used CsCl/EtBr method. I think you should choose the appropriate method depends on your purpose."

I could not attach the file here, but please check this link, http://jb.asm.org/content/145/3/1365.abstract. Hope this helps.

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4/11/2012 11:15:22 AM  

i need help in consrructing a flow chart for the isolation of bacterial DNA(E.coli)

Fanglian

4/12/2012 9:10:46 AM  

Here is the flow chart for this protocol: grow cell culture--spin down the cell--resuspend the cell pellet with P1 buffer--lyse cells with P2 buffer--neutralize lysis solution with P3 --spin down to discard insoluble chromosomal DNA, proteins & large RNA molecules, and save the supernatant--precipitate plasmid DNA by ethanol--dissolve DNA in TE buffer or sterile water.

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4/12/2012 1:43:23 AM  

what's the principle of the isolation of bacterial plasmid?

Fanglian

4/12/2012 9:12:28 AM  

here is a great reference for your question,
Birnboim H.C., Doly J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6): 1513-23.

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4/21/2012 6:59:26 PM  

Function of three solution.

Fanglian

4/23/2012 1:55:02 PM  

please see this reference, Birnboim H.C., Doly J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6): 1513-23.

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11/9/2012 12:39:15 AM  

Hello.
why after adding solution2 our solution became viscoses?

Fanglian

11/27/2012 6:29:23 AM  

Yes, after adding the lysis buffer,the suspension does become viscous, which may due to the presence of genomic DNA.

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12/27/2012 2:24:56 AM  

What is the purpose of ethanol in washing buffer?

Fanglian

1/1/2013 1:16:27 PM  

I am not sure which wash buffer was asked. I assumed you meant ethanol in step 9 of procedure, which is to percipitate DNA.

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devidayal

5/23/2013 7:08:13 AM  

what activity when we add.Ammonium acetate + propane-1-ol rather then 100% ethanol .

Fanglian

8/17/2013 1:02:25 PM  

I apologize for my delay in response to your question.
Ammonium acetate, like sodium acetate, can neutralize the charge on the sugar-phosphate backbone of the DNA, which can help DNA precipitation. Ethanol only always works to me that is why I did not try other ways to precipitate DNA. But, there are many good references about this topic, such as http://bio.wayne.edu/profhtml/Cunningham/private/privatedocuments/EtOH.pdf

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Cherysa

6/19/2013 4:22:19 PM  

Hello,

Do you have any suggestion for plasmid extraction from staphylococcus aureus. Also do you have any protocols for transformation of staphylococcus aureus other than via electroproration?

thank you.

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cecilia

7/4/2013 6:42:24 AM  

WHAT IS THE CONCENTRATION OF ICE COLD BUFFERED SODIUM ACETATE TO BE USED IN PLASMID EXTRACTION BY BRINBOIN AND DOLLY(1979)

Fanglian

8/17/2013 1:17:22 PM  

In the paper (BRINBOIN AND DOLLY,1979), sodium acetate with different concentrations was used at several different steps. So, Please read it (using the link below) by yourself for your specific question.
http://www.its.caltech.edu/~ctobin/miniprep/Birnboim,%20Doly.pdf

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Neha

8/16/2013 7:45:28 AM  

HELLO,
I m a student working on a virus. I used to isolate the plasmid using the alkali lysis method but i don't get the pellet. Can u tell me the possible reasons behind it. Please help me.

Fanglian

8/17/2013 1:26:03 PM  

Is your plasmid low copy or high copy? As mentioned in this protocol, it works well to plasmid with > 10 copies. If it is lower copy, you can use more bacterial culture and scale up the volume of each buffer solution accordingly. Another way to increase the yield, as mentioned in step 9, keep your tube at -20 degree overnight before spinning.
Hope this will help.

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Rina

10/11/2013 9:55:00 AM  

What is the meaning of 200- proof cold ethanol

Fanglian

10/11/2013 10:08:08 AM  

It is the same as 100% ethanol (pure ethanol). For the exact meaning of ethanol proof, please check it out at http://en.wikipedia.org/wiki/Alcoholic_proof

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Rina

10/12/2013 12:42:16 AM  

We are getting pallets bt after observing under Gel dog DNA bands are not observed. What will be the reason behind this?

Fanglian

10/13/2013 4:31:46 PM  

1. How much the DNA pellet is suspended? Could it be too diluted? Try to load more to the DNA gel.
2. It might get degraded.

Rina

10/13/2013 6:28:36 PM  

Thanks ........ I will try that...

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Rina

10/17/2013 2:12:34 AM  

Why we icubate at -20°c during RNA isolation?

Fanglian

10/30/2013 3:07:46 PM  

Sorry for my delay in relying. Did you mean "-20 °C" at step 9 in the above protocol? It is supposed to be helpful for DNA precipitation. Although it was also recommended to keep it on wet ice, I never try it.

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golam

1/16/2014 9:32:46 AM  

Hi thanks for nice protocol

Is it possible to isolate original plasmid DNA from E.coli by this method. Actually I mean simply by culturing the E. coli cell without transferring any plasmid extrnally.

Thanks

Fanglian

1/17/2014 5:09:08 PM  

Sorry I do not know the answer since I never tried that. Please let us know the outcome if you would try. Thanks!


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SANJEET

1/21/2014 11:27:21 PM  

Why are we using ethanol of two different concentration(100% ethanol in DNA precipitation and 70% while discarding the supernatant) in this method? Can't we use only one?

Fanglian

1/24/2014 5:39:54 PM  

At step 9 above, you have to use 100% ethanol, and the final concentration of ethanol in the solution is about 75%. In my protocol (step 11), I did not use 70% ethanol to wash the DNA pellet, and it worked in my hand. Of course, you can add the extra wash step with 70% ethanol.

Hope this helps.

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riz

2/6/2014 9:18:04 PM  

why don't you use lysozyme in solution 1. I extracted plasmid DNA using lysozyme in solution 1 and after step 6 I added an equal volume of phenol:chloroform to remove protein. If I follow this method will I be able to remove protein?
After extracting the DNA I ran it on a gel. But the extracted DNA was fragmented and the average size was about 300 bp. I'm not sure what went wrong. Can you please advice me to overcome this problem.

Fanglian

2/11/2014 7:24:34 PM  

I did not use lysozyme for E. coli cells. Maybe you need the enzyme to break down other bacterial species. Step 6 should be able to remove proteins. While additional phenol:chloroform step helps to remove remaining proteins, if you need high purity of DNA, I would suggest that you use some commercial plasmid extraction kits.

In any case, both of your modifications (addition of lysozyme and phenol:chloroform step) should not be the cause of the DNA degradation.

Two things I can come up with at this moment to avoid the problem: 1). do not vortex the tubes after step 4 above. 2). please make sure that all reagents/ solutions (including water), tubes, pipette tips need to be DNAase-free.

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wang

5/14/2014 5:40:59 PM  

I have extracted plasmids DNA according above protocol with 8ml culture.The concentration of dsDNA is about 4000ng/ul,however electrophoresis band is weak.I think there are too many RNAs ,how to deal with it?

Fanglian

5/19/2014 9:52:39 AM  

Please see the note at step 12 above.

Good luck,
Fanglian

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Nguyen

5/15/2014 3:26:49 AM  

Hi,
I am going to extract Bacteriophage from soil. Could you suggest me any kind of method or protocol?
Thanks

Fanglian

5/19/2014 10:28:03 AM  

The following one may be interesting to you:

"Isolating RNA from the Soil" by Jacqueline M Chaparro and Jorge M Vivanco at http://www.bio-protocol.org/wenzhang.aspx?id=903

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ani

7/21/2014 10:13:08 AM  

why gDNA is not isolated with pDNA?

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ani

7/21/2014 10:14:56 AM  

how to get rid of gDNA contamination with pDNA?

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ani

7/21/2014 10:19:12 AM  

why plasmids are used as a tool for gene transfer?
differenciate viral DNA and pDNA?
name 10 plasmids and its host organism?

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krishna

9/5/2014 9:17:56 AM  

Hi,
how to get rid off chopped RNA from final solution >?

Fanglian

9/10/2014 2:58:01 PM  

As suggested in the note of step 12, RNAase can be used to remove the RNA contamination.

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maham

9/7/2014 10:38:29 AM  

why it is written as 'test tubes', as the whole culture was transferred in just one tube.

Fanglian

9/10/2014 2:56:41 PM  

Yes. But, as indicated in step 2, for 3 ml O/N culture, need to spin down twice (transfer 1.5 ml each time to the Eppendorf tube). Hope it is clear to you now.

--Fanglian

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MUHAMMAD

10/8/2014 9:36:03 AM  

WHY HERE NOT USING ACETIC ACID FOR NEUTRALIZING SOLUTION INSTEAD OF POTASSIUM ACETATE EVEN THOUGH BOTH HAVE SIMILAR pH ?

Fanglian

3/7/2015 4:46:25 PM  

That is because potassium acetate, but not acetic acid can be used as a buffer.

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MUHAMMAD

10/8/2014 9:42:54 AM  

WHAT IS LOW COPY AND HIGH COPY?

Fanglian

3/7/2015 4:52:21 PM  

"A plasmid may be present in an individual cell in varying number, ranging from one to several hundreds. The normal number of copies of plasmid that may be found in a single cell is called the copy number, and is determined by how the replication initiation is regulated and the size of the molecule. Larger plasmids tend to have lower copy number.[7] Low copy number plasmids that exist only as one or a few copies in each bacterium..." ( http://en.wikipedia.org/wiki/Plasmid)

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ILANGO

4/22/2015 10:01:35 PM  

Hi,

Our strain is recombinant E.coli with tetracycline marker,we need to plasmid isolation for this step the initial growing media with LB broth/Agar OR Our own developed chemical defined media,please explain to me.

Thank you

regards
Ilango

Fanglian

4/22/2015 10:36:31 PM  

Hi Ilango,

Sorry I am not sure your question. Were you asking if you should grow your strain in LB media or in the medium that you developed by yourself? If yes, below is my answer to your question:

There is no constraint on the choice of growth medium in this protocol. If your strain grows happily in both mentioned media (with addition of antibiotics to select for the plasmid of your interest), I think you can use either of the media to grow the cells that isolate the plasmid.

Hope your question was answered above.

--Fanglian

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saeed

7/12/2015 10:01:03 PM  

can you tell me how much concentration of plasmid DNA is required for cloning? and how we will increase the Plasmid DNA concentration?

Fanglian

7/20/2015 12:08:32 PM  

Hi Saeed,

Regarding your 1st question, you can find the answer from another protocol at http://www.bio-protocol.org/e52

Regarding your 2nd question, add 2.5-3 volume of 200-proof cold ethanol (pre-cool at -20 °C) to your DNA sample. Also, add 1 μl glycogen (1 μg/μl, e.g. Roche, catalog number: 10901393001) to aid precipitation. Mix them by inverting the tubes for a few times. Keep the tube at -20 degree for at least 30 min before spinning down plasmid DNA at highest speed for 10 min at 4 degree. The, wash pellets with 150 μl 70% ethanol. Let the pellet air dry before adding small vol. of your DNA suspension solution.

Good luck,

--Fanglian

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sushil

8/4/2015 1:50:05 AM  

respected madam/ sir you site nice , experimental protocols are too good , i hv simple question to ask you please can tell me is there any random method to indentify the , presence or absence of plasmid in any bacterial cell,

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