Abstract
This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits. This technique was invented by Birnboim and Doly (1979).
Materials and Reagents
Equipment
Procedure
Note: All steps except of steps 9 and 10 are carried out at RT.
Recipes
Acknowledgments
This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hello, the working concentration that was used is 1 mg ml-1 (1-5uL) of RNAase
Buy it as is
That's because during the renaturation step ie, during addition of solution III (Pot. acetate solution), smaller plasmid DNA renatures fast but long chromosomal DNA cannot. Also these single stranded DNAs along with other residual protein parts due to hydrophobic interactions coagulate to form a white precipate which is pelleted out after this step.
Any other organisms could be used. This particular E.coli strain is used because of its availabilty, non-pathogenicity and mainly due to its antibiotic susceptible nature, which would make the transformants selection process more efficient.
Hi Saeed, Regarding your 1st question, you can find the answer from another protocol at http://www.bio-protocol.org/e52Regarding 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
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
"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)
That is because potassium acetate, but not acetic acid can be used as a buffer.
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
As suggested in the note of step 12, RNAase can be used to remove the RNA contamination.
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
Please see the note at step 12 above.Good luck,Fanglian
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.
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.
Sorry I do not know the answer since I never tried that. Please let us know the outcome if you would try. Thanks!
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.
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.
Thanks ........ I will try that...
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
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.
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
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
I am not sure which wash buffer was asked. I assumed you meant ethanol in step 9 of procedure, which is to percipitate DNA.
Yes, after adding the lysis buffer,the suspension does become viscous, which may due to the presence of genomic DNA.
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.
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.
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.
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.
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.
All steps except of steps 9 and 10 are carried out at RT.
Because RNase is active at normal room temperature, better results can get if you do all the steps, using ice bucket is also sufficient.
Better do all the steps at cold conditions, and prepare all the recipies in DEPC treated water only for good yield of plasmid.