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This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.

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[Bio101] E. coli Genomic DNA Extraction Updated version
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Microbiology > Microbial genetics > DNA
Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
7/20/2011, 70473 views, 46 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.97

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.

Materials and Reagents

  1. Tris base (Calbiochem-Behring)
  2. Proteinase K (Sigma-Aldrich)
  3. Phenol\chloroform (1: 1) (EM Science)
  4. 200 proof ethanol (Pharmco-AAPER)
  5. RNAase (Life Technologies, Invitrogen™)
  6. Ethanol
  7. SDS
  8. EDTA
  9. Tryptone
  10. Yeast extract
  11. NaCl
  12. LB medium (see Recipes)
  13. TE buffer (see Recipes)
  14. Lysis buffer (see Recipes)

Equipment

  1. Tabletop centrifuge (Eppendorf)
  2. 1.5 ml Eppendorf tube
  3. Incubator
  4. Gloves

Procedure

  1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
  2. Discard the supernatant.
    Note: Remove as much of the supernatant as you can without disturbing the cell pellet.
  3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.
  4. Incubate 1 h at 37 °C.
  5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
    Note:
    Do not vertex the tube—it can shear the DNA.
  6. CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.
  7. Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
  8. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
  9. Steps 4-6 can be repeated until the white protein layer disappears.
  10. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
  11. Spin at max speed for 5 min.
  12. Remove aqueous layer to new tube.
  13. To precipitate the DNA, add 2.5 or 3 volume of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
    Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see DNA pellet. It looks transparency when it is wet and turns to white when it becomes dry.
  14. Incubate the tube at -20 °C for 30 min or more.
  15. Spin at max speed for 15 min at 4 °C.
  16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
  17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
  18. Resuspend DNA in TE buffer.
    Note: Large amounts of RNA will be present in the DNA sample. So, 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 lysis buffer with a final concentration of 1 mg ml-1.
  19. Check isolated Gemonic DNA on an agarose gel.
    Note: we expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded.

Recipes

  1. LB medium
    1% tryptone
    0.5% yeast extract
    200 mM NaCl
  2. TE buffer
    10 mM Tris-Cl (pH 8.0)
    1 mM EDTA (pH 8.0)
  3. Lysis buffer (10 ml)
    9.34 ml TE buffer
    600 ul of 10% SDS
    60 μl of proteinase K (20 mg ml-1)

Acknowledgments

This protocol was adapted from Andrew Binns’ lab protocol collections at the University of Pennsylvania (USA).

References

  1. Maniatis T., E.F. Fritsch, and J. Sambrook (1982). Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.


How to cite this protocol: He, F. (2011). E. coli Genomic DNA Extraction. Bio-protocol Bio101: e97. DOI: 10.21769/BioProtoc.97; Full Text



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10/21/2016 4:08:12 PM  

Henry Wong
Universiti Tunku Abdul Rahman

Hi.

(i) May I know how long do you store for the leftover lysis buffer?
(ii) Will it be fine if I use MH broth instead of LB broth?
(iii) How much TE buffer do you add for step 18?
(iv) If I were to determine the concentration of DNA eventually using spectrophotometry, how can I get the dilution factor for the amount of TE buffer added in (iii)?
Thank you in advance for the respond and providing the protocol.

The latest modification time: 10/21/2016 4:16:54 PM

10/22/2016 1:41:15 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Henry,

Please see my responses to each of your questions below.

(i) May I know how long do you store for the leftover lysis buffer?

Proteinase K has to be added freshly. Thus, lysis buffer containing proteinase K should not be used next day. Otherwise, it can be kept at room temperature at least 6 months.

(ii) Will it be fine if I use MH broth instead of LB broth?

I never tried MH broth. But, I think it should be fine if your bugs grow happily in the media.

(iii) How much TE buffer do you add for step 18?

50 microliter.
(iv) If I were to determine the concentration of DNA eventually using spectrophotometry, how can I get the dilution factor for the amount of TE buffer added in (iii)?

Well, depending on how you would do the dilution, I guess you have to do your own math to get the answer.

--Fanglian

10/24/2016 3:08:05 PM  

Henry Wong
Universiti Tunku Abdul Rahman

Thank you Fanglian. Another two questions:
i. How do you ensure that the overnight culture of E. coli is sufficient to provide you the yield between 5 to 10 micrograms?
ii. If I plan not to add in my RNAase into the lysis buffer, do I add RNAase after the step of resuspending the DNA in TE buffer right before checking it on an agarose gel? How long do I need for the incubation?

The latest modification time: 10/24/2016 3:08:51 PM

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9/26/2016 11:19:11 AM  

varsha wagh
NIPER AHMEDABAD

Hi,i got good yield of plasmid DNA after RNase treatment but after purification with phenol- chloroform:isoamyl alcohol the yield decreased.what can be the reasons behind this.thank you

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8/11/2016 7:33:00 AM  

ku dyyla
University Malaysia Kelantan

Hai. I want to ask, how many the volume of RNase should i added into my lysis buffer. Because i want to remove the RNA.
Sorry i can understand the explaination in the note on step no8

8/11/2016 11:06:57 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Ku,

Depending on the concentration of your RNAase stock, a final concentration of RNAase in your lysis buffer is about 1 mg/ml.

Hope this helps.

Good luck,
Fanglian

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5/11/2016 5:30:55 AM  

samaila waziri
Ahmadu Bello University, Zaria, Kaduna State, Nigeria

dear Fanglian, I plan on taking a study on the detection of E. coli in groundwater samples. I need a E. coli DNA extraction and amplification procedure that does not require the use of Kit. you could post the methodology/procedure to my e-mail address: smlbnjmn@gmail.com

5/11/2016 6:15:02 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Samaila,

Please feel free to save a copy of the above protocol at your end.

Good luck,
Fanglian

5/18/2016 11:37:17 AM  

samaila waziri
Ahmadu Bello University, Zaria, Kaduna State, Nigeria

dear fanglian, I will also require a detailed procedure for the detection of E coli in water samples that require a DNA extraction Kit and PCR mastermix

5/20/2016 9:21:56 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

There are a number of companies (i.e. Qiagen) providing the related commercial products with detailed manufacture's instructions. Thus, Bio-protocol does not publish this type of protocols as a separate publication. You could either find the instructions on the company websites or contact them directly.

Good luck!
Fanglian

5/21/2016 10:54:33 PM  

samaila waziri
Ahmadu Bello University, Zaria, Kaduna State, Nigeria

thank you fanglian

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5/11/2016 5:30:09 AM  

samaila waziri
Ahmadu Bello University, Zaria, Kaduna State, Nigeria

dear Fanglian, I plan on taking a study on the detection of E. coli in groundwater samples. I need a E. coli DNA extraction and amplification procedure that does not require the use of Kit. you could post the methodology/procedure to my e-mail address: smlbnjmn@gmail.com

Reply

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4/4/2016 9:30:03 AM  

Anthony Bayega
McGill University

Hi, thanks for the protocol. Does this protocol yield high molecular weight DNA? I am interested in extracting like 80kb and above. Also, after the lysis step, is the lysate not too thick (mucous) to handle and even seperate the proteins out after phenol-chloroform?

4/5/2016 6:08:07 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Anthony,

Yes, you should expect to see DNA with high MW by using this protocol. And, the lysate was not too thick to handle in my hands.

Good luck!
Fanglian

4/6/2016 8:59:56 AM  

Anthony Bayega
McGill University

I am running the protocol now and will let u know the results. Thanks

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8/24/2015 10:42:59 PM  

upasana pathak
hnmrs

HELLO,
could you suggest a protocol for S.aureus genomic DNA extraction or for gram positive bacteria..i have been carrying out dna extraction by the sodium perchlorate metthod but lately there is lot of white precitate which i get and dna does not get extracted

8/25/2015 2:47:45 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,

I will contact one of our authors who works on S. aureus about your request and let you know when I receive his response.

--Fanglian

8/26/2015 9:46:36 PM  

upasana pathak
hnmrs

hey,thankyou I will be waiting

8/26/2015 10:58:21 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi, Upasana,

I have asked Prof. Chikara Kaito at the University of Tokyo for help with your question. Below is his response:

"The extracting protocol of S. aureus genomic DNA has been written in Novick RP, Methods in Enzymology, Vol 204, 587-636.
The method is written on page 589-590.

For convenience, we are usually using the extraction Kit from Qiagen (QIAamp DNA Blood Mini Kit, Cat No. 51106) after lysing S. aureus cells with lysostaphin."

Hope this would be helpful. Here, I want to thank Prof. Chikara Kaito again for his prompt response and helpful information.

Good luck,

--Fanglian

The latest modification time: 8/26/2015 10:58:45 PM

8/27/2015 9:48:32 PM  

upasana pathak
hnmrs

hello,
thankyou very much for the reference..i also had one doubt when we precipitate DNA with cold ethanol i see very nice precipitate as soon as etanol is added but which disappears on shaking or even slight mixing..what is the probable reason??

8/27/2015 10:10:06 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,

It may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down. You should see DNA pellet (it looks transparency when it is wet, and turns to white when it becomes dry) .
--Fanglian

The latest modification time: 8/27/2015 10:10:16 PM

10/21/2015 11:03:36 PM  

humera tariq
university of lahore

hi ,
which DNA ladder was run in above image with genomic DNA?
how much time is required to migrate the genomic DNA at least from the well?

10/26/2015 12:30:14 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi, Humera,

Regarding the DNA ladder, please contact the company via the provided link above. Regarding your 2nd question, unfortunately, I did not record the time when I ran my genomic DNA from E coli. But, my impression was that it should not take too long. In case your sample was too condensed, you might run your samples with 1 or 2 more dilutions.

--Fanglian

The latest modification time: 10/26/2015 12:30:51 PM

11/3/2015 8:52:11 PM  

humera tariq
university of lahore

thanks Fanglian:)

2/24/2016 10:31:38 AM  

Karthikeyan RK
Shanmuga

How can isolate The DNA from onions. Please help to me

3/1/2016 10:56:05 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Karthikeyan,

Sorry, I 've never done that experiment. Please use Bio-protocol RaP service (http://www.bio-protocol.org/RaP.aspx) to request such a protocol.

Thanks,
Fanglian

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7/31/2015 1:18:28 PM  

tebogo masedi
university of limpopo

what are the results expected for the isolation of DNA from E.coli

The latest modification time: 7/31/2015 1:19:31 PM

7/31/2015 2:06:14 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi, sorry I do not have the requested image to show at this moment. But, I found a good example from this link, http://www.mobio.com/microbial-dna-isolation/powermag-microbial-dna-isolation-kit.html (Figure 2 is copied below)

 

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7/23/2015 6:55:21 AM  

Rachel Williams
University of southampton

Hey, i've been searching for a solid genomic DNA extraction protocol to follow for an transformed E.coli culture. The main difference i've noticed is the call for CTAB. Clearly the process can work without but do you know how beneficial it is to use? Thanks!

7/23/2015 10:05:09 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Rachel,

What does CTAB stand for? Any references?

Thanks,
Fanglian

7/24/2015 12:45:24 AM  

Rachel Williams
University of southampton

hexadecyltrimethyl ammonium bromide
I've seen it in a few protocols such as this one:
http://1ofdmq2n8tc36m6i46scovo2e.wpengine.netdna-cdn.com/wp-content/uploads/2014/02/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf

7/31/2015 2:01:14 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Thank you for sharing, Rachel!
From the original paper about CTAB method by Kate Wilson (http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0204s56/abstract), it seems that CTAB can help to remove polysaccharide contamination in DNA prep. This would be useful since "exopolysaccharides can interfere with the activity of enzymes such as restriction endonucleases and ligases." (quoted from the original paper).

The latest modification time: 7/31/2015 2:10:13 PM

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6/16/2015 10:10:03 PM  

Pushpalatha.R pushpa
GKVK

why Glucose/Sucrose is added during plasmid DNA isolation and not during genomic DNA?

6/20/2015 12:57:35 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,

For the protocol "Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method", Glucose is added (in resuspension solution) to make the solution isotonic. In fact, isotonicity is not required for bacteria with cell walls (i.e. E. coli) because bacteria with cell walls can withstand wide range of solution concentration. Thus, glucose may not have to be included in the resuspension buffer. I never tried the one without glucose, though. So, please share your experience with us if you try it without glucose.

Good luck,
Fanglian

6/20/2015 10:29:40 PM  

Pushpalatha.R pushpa
GKVK

Even I tv not tried so. In reference point of view. Few protocols shows use of glucose/sucrose for plasmid DNA extraction and not been used during genomic DNA isolation of a bacteria. Why so...

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4/24/2015 11:44:12 PM  

oji pku
student

the obtained DNA sample from bacterium is stored in TE buffer (10 mM Tris, pH 7.6 and 1 mM EDTA). Upon addition of 100 microliter of ethanol, no precipitate is seen. why not? what do I do to recover the DNA? Thank you

4/25/2015 12:25:10 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,

Did you obtain the DNA sample by using this protocol? In this protocol, TE is added to resuspend the DNA pellet after ethanol precipitation.

In any case, to precipitate your DNA, you need to make sure DNA : (absolute) ethanol = 1 vol: 2.5-3 vol (ethanol needs to be pre-cooled at -20 degree). Also, you can add 1 μl glycogen (1 μg/μl) to aid precipitation.

Hope this helps.

Good luck,
Fanglian

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4/12/2015 11:33:39 PM  

他能提取大肠杆菌的整个DNA基因组吗?

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10/21/2014 6:05:47 PM  

Nguyen Hoa
University of Science

can I ask question? If we use RNAse in the step 7, incubate with the protease and SDS. Will the protease or SDS denature or degrade my RNAse. I think that use the phenol/chloroform to get rid of protease and then incubete with RNAase then repeate again with phenol/ chloroform to eliminate the RNAse.
Thanks.

10/27/2014 2:53:03 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi, Nguyen,

You can inactivate protease K at 95 °C for 15 min.

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9/19/2014 5:37:30 AM  

Wang Gui
WUHAN University

Hi,
Can I use this protocol to extract a mass of genomic DNA? Should all of the solutions were scaled increased?

Thanks in advance!

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9/19/2014 5:35:41 AM  

Wang Gui
WUHAN University

Hi,
Can I use this protocol to extract a mass of genomic DNA? Should all of the solutions were scaled increased?

Thanks in advance!

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9/19/2014 5:35:09 AM  

Wang Gui
WUHAN University

Hi,
Can I use this protocol to extract a mass of genomic DNA? Should all of the solutions were scaled increased?

Thanks in advance!

10/27/2014 2:44:48 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Never did. But, your suggestion sounds reasonable to me.

10/28/2014 12:45:05 AM  

Wang Gui
WUHAN University

Thanks all the same.

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9/19/2014 5:22:39 AM  

Wang Gui
WUHAN University

Hi,
Can I use this protocol to extract a mass of genomic DNA? Should all of the solutions were scaled increased?

Thanks in advance!

Reply

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9/19/2014 5:22:04 AM  

Wang Gui
WUHAN University

Hi,
Can I use this protocol to extract a mass of genomic DNA? Should all of the solutions were scaled increased?

Thanks in advance!

Reply

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9/7/2014 11:19:53 AM  

maham khan
uog

marvellous

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7/16/2014 11:00:53 AM  

prasanna kumar
JNTU K

in which phase(top or bottom) the lysis buffer will be present after done with addition of phenol and choloroform and centrifuging? during 10th step?

7/23/2014 2:39:28 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,
It is on the top (that is "the upper aqueous phase" as stated in 10th step), not the bottom layer.

--Fanglian

7/23/2014 7:37:17 PM  

prasanna kumar
JNTU K

but the molecular weight of lysis buffer is more than the bottom phase that contains isoamyl alchol and cholorofrom, when we have done with centrifugation, high molec wegith compounds will settled down, right, so i'm in confusion with that phase formations madam


regards & thank you

prasanna kuamr T

8/11/2014 11:41:29 PM  

suresh Babu
Bionary BioProducts PVt LTd

Hi Fanglian,

I have small query in the protocol u have mentioned,
To precipitate the DNA we need to add 200 proof ethanol to the aqueous layer taken in the new tube in the 14th step u mentioned?
Small confusion explain me.

Thanks in advance
Suresh G

10/27/2014 2:42:43 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Suresh,

Yes, as described at step 15, 2.5-3 vol of 200 proof ethanol (stored at -20 C) is added to the aqueous layer taken in the new tube from step 14.

Hope your question is clarified.

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6/13/2014 4:41:52 PM  

Kin Pong U
UCL

Hi Fanglian,

May I ask will this protocol work if I want to isolate bacterial genomic DNA together with plasmid DNA?

Kind Regards
Ken

6/13/2014 5:46:41 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Ken,

Good question! While I never tried it, theoretically I think it should work. If you try it with a high-copy plasmid, you may be able to distinguish plasmid DNA from genomic DNA on a DNA gel if it works.

6/13/2014 5:53:41 PM  

Kin Pong U
UCL

Hi Fanglian,

Thank you very much.

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3/31/2014 8:05:49 AM  

neha kakade
stuent

what is the fuction of lysis solution without SDS
LYSIS SOLUTION WITH SDS.IN GENOMIC DNA

3/31/2014 10:03:28 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Neha,

lysis buffer needs SDS or other detergents (like Triton x-100) to disrupt cell membranes.

--Fanglian

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3/31/2014 8:02:56 AM  

neha kakade
stuent

HI..... mam can i ask one question

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3/12/2014 9:32:26 PM  

Hansul Jung
Korea University

I did this experiment but my figure was very low. I am just student who has studied this part and this experiment i did was my first experiment. My teacher said over 500ng/ul is proper outcome of this experimnet. Some people got a over 100ng/ul figure. Why did i take it like this ?


AU 230 0.04
AU 260 0.09
AU 280 0.09

260/280 2.10
260/280 1.01

ng/ul 4.32

3/13/2014 2:21:31 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Hansul,

I am afraid that your DNA sample got lost or degraded somehow. Please see my comments on 8/17/2013 and 2/3/2012 below. Hope you would find them helpful.

Good luck,
Fanglian

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3/4/2014 1:53:47 PM  

Mark Lostonery
INCA

How can I separate genomic DNA from carbohydrate. There seems to be a lot of carbohydrate in the prep.
thanks

3/10/2014 4:54:20 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Mark,

I'd agree with you. But, I never tried to remove the carbohydrate contamination from my genomic DNA prep. I think you may have to use some commercial kits to get more pure genomic DNA.

Good luck,
Fanglian

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1/15/2014 12:40:48 AM  

seph richards
masinde muliro university of science and technilogy

in E. coli genomic DNA extraction,why use Tris base

1/17/2014 5:21:23 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Tris-Cl/Tris-HCl in TE buffer needs Tris base. The pH is adjusted to 8.0 by HCl.

5/11/2014 10:33:51 PM  

seph richards
masinde muliro university of science and technilogy

is protocol for RNA extraction from the soil published online already!?

5/13/2014 1:25:48 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Seph,

Yes, please see "http://bio-protocol.org/wenzhang.aspx?id=903". Actually you can find it by searching some key words, like "RNA, soil" (in this case) on the Bio-protocol home page.

Thanks,
Fanglian

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11/8/2013 12:43:56 PM  

bio 22
facultyof science

Prepare 500ml of TE25S buffer which consists of 25mM Tris-HCL pH8.0, 25mM EDTA pH8.0 and 0.3 M sucrose.
If you have:
1M Tris-HCL
0.5 M EDTA
M.wt of sucrose=?342.29648 g/mol

11/12/2013 2:13:55 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

I believe you could figure it out if you give yourself some more time.

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10/29/2013 12:31:32 PM  

wessam bio
college

WE HAVE USED VERY STRONG ORGANIC SOLVENTS TO EXTRACT DNA >WHY IS DNA UNAFFECTED BY YHESE SOLVENTS?

10/31/2013 1:00:51 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi,

Phenol/chloroform, used in the above protocol are used to denature proteins , not nucleic acids. However, oxidized phenol can damage the nucleic acids. For some related references, you may find it at http://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extraction.

--Fanglian

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10/28/2013 7:42:33 AM  

zeeshan saiyed
srki

how to carry out calculation .to know the purity of genomic dna isolated from E.coli.

10/28/2013 12:58:09 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

You can quickly get some idea (not accurate estimation, though) about the purity of genomic DNA by the following three ways:

1.For checking protein contamination, you can take the reading of A260nm/A280nm. If A260nm/A280nm is about 1.8-2.0, it indicates that your DNA sample is quite pure--not much protein contamination.

2.For checking contamination of some organic compounds or salts (phenol would be the main source of this type of contamination by using the above protocol), take the reading of A260nm/A230nm. For a good DNA prep sample,A260nm/A230nm is about 1.5-1.8.

3. For checking RNA contamination, you can check it on a DNA gel. As mentioned in the protocol, RNA contamination can be easily removed by RNase.

Please see below for some recommended references related to above suggestions:
1. Maniatis T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
2. http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf

--Fanglian

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9/5/2013 12:06:15 AM  

tamil selvi
asan memorial college

HOW TO KNOW THE DIFFERENCE BETWEEN THE DNA, RNA AND PLASMID BAND ON THE AGAROSE GEL UNDER UV

9/8/2013 2:18:29 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

You may be able to tell the difference based on their patterns and sizes. For genomic DNA, most of them are very big and do not migrate far from well. And, since they often consist of DNA fragments with different sizes, you can bands with the big range of sizes (bands are smear if many got degraded).
For plasmid DNA, when they are uncut, they often appear more than one band on the gel (due to different topology). To confirm the plasmid DNA of your interest, you can cut the DNA with known restriction enzymes before run the gel.
For RNA, I do not have much experience on running specific RNA samples on agarose gels (may need denaturing agarose gel). But, for RNA contamination found in your DNA prep (without RNase treatment), RNA bands (often very bright) migrate toward the bottom of the gel.
Hope this could be helpful.

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8/31/2013 10:55:27 AM  

Palak Parashar
Delhi University

why we use e.coli for plasmid dna extraction, why not any other bacteria

8/31/2013 11:32:06 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

While we often extract plasmid DNA from E. coli, people do extract plasmid DNA from other bacteria depending on their study purposes. The following are some reasons why we often use E. coli to extract plasmid DNA.

1. E. coli is one of the most well-established model organisms for bacteriology.
2. Since protein expression and purification in E. coli is very easy and efficient, it is one of the most commonly used prokaryotic expression systems.
3. Many commercial and non-commercial plasmids/expression vectors have been made for E. coli.

Hope my answers make sense to you.

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8/26/2013 2:27:22 AM  

sivasankaran mrm
madras university

HELLO THERE,
NEED OF PROTOCOL FOR WHOLE GENOME EXTRACTION FROM SOIL SAMPLE
THANK YOU

8/26/2013 9:49:57 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Sorry that we do not have that protocol at this moment.

8/28/2013 10:43:58 PM  

sivasankaran mrm
madras university


no problem and thank you for the reply . if any related protocol is available for whole gemome extraction would help me.

8/31/2013 11:36:23 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

We will have a protocol "Isolating RNA from the Soil" Jorge Vivanco group (Colorado State University) published online next month (Sept. 20th), which may be interesting to you.

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8/17/2013 11:56:37 AM  

gugu ndiema
ukzn westvile

hey.........can I ask something?

8/17/2013 12:47:25 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

sure

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5/2/2013 5:30:25 AM  

Pooja verma
university of delhi

what is the use of NaCl?

8/17/2013 12:21:59 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Sorry for my delayed response. Which NaCl (from which buffer/solution) you were asking?

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2/20/2013 3:20:10 AM  

Thabeng Thabeng
University of Botswana

outline to me the possible explaination or disscussion when no DNA bands were not seen in gel electrophoresis!!thanxs in advance

8/17/2013 12:47:01 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Please see below for some possible explanations:
1.You could easily get no DNA if you did not carefully/exactly follow each step of the protocol.
2. Your DNA may got degraded (see my answers for this issue posted on 2/3/2012 below)
3. As mentioned at step 15 above, you should be able to see (try to see the tube against light)DNA precipitation (white color)floating in the solution after mixed with cold ethanol DNA precipitation. If not, that means you have lost the DNA before that. If you do see it, you can keep the tube at -20 degree overnight, which will increase the yield. Another way to increase the yield is that you could use more cell culture (e.g. 5 ml).
Good luck!

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1/28/2013 3:25:06 AM  

"So, for subsequent reaction, for example, to digest plasmid DNA, add 1-5 ul (1 mg/ml) RNAase to the digestion solution to completely remove RNA" I don't understand... Why RNAase could digest plamsidic DNA? Thanks!

1/29/2013 12:54:00 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

It is typo...It should be" to digest/cut genomic DNA (for construction of library)". Sorry about confusion. But, RNase is used to digest RNA contamination in genomic DNA prep.

1/29/2013 12:54:00 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

It is typo...It should be" to digest/cut genomic DNA (for construction of library)". Sorry about confusion. But, RNase is used to digest RNA contamination in genomic DNA prep.

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1/5/2013 5:49:12 AM  

why 260/280 we do

1/12/2013 12:46:05 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Ab260nm/280nm ratio indicates the purity of DNA (or RNA) and level of protein contamination in your DNA/RNA sample. 260/280=2 indicates 100%DNA.

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11/27/2012 8:30:15 PM  

Not getting results in transformed ecoli

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11/27/2012 7:03:56 AM  

why cant we use NaCl instead of NaOAc?

12/16/2012 7:47:28 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Acidity of sodium acetate can neutralize the charge on the sugar-phosphate backbone of the DNA, which can help DNA precipitation.

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11/25/2012 10:20:04 AM  

rekha singh
school of biotechnology DAVV Indore India

what is the role of the sodium acetateis....??

11/27/2012 6:48:10 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

High concentration of sodium acetate (3M)can be used to precipitate high molecular weight molecules including genomic DNA. I did not use it in my experiments. But, it might help to increase the DNA yield by using sodium acetate.

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11/13/2012 1:03:26 AM  

Hello! what is the yield of the Isolated DNA???

11/27/2012 7:10:57 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

For 1.5ml overnight cell culture, typically I can obtain 5-10 ug DNA.

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8/24/2012 3:19:03 AM  

there is any alternatives of proteinase K ?

8/24/2012 12:25:00 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Sorry I did not try anything else since proteinase K is always available in lab.

7/29/2013 6:33:40 PM  

mourad ali
scinetest

also we use EDTA as an alternative of proteinase k with high concentration from 20mM to 30mM
^^

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8/24/2012 3:15:41 AM  

is it not any other protocol for extraction of dna from ecoli without proteinase K

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6/3/2012 12:24:17 PM  

can you please state the reasons for adding each and every reagent?

6/12/2012 11:42:49 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

In lysis buffer, proteinase K is to digest proteins including membrane proteins, and SDS facilitates digestion of cells by denaturing and solubilizing membrane proteins. EDTA is used to to inhibit DNases. Phenol/chloroform is used to remove proteins from DNA sample. Ethanol is used to precipitate DNA.

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4/25/2012 3:53:12 AM  

CAN U PLACE SHOW SOME RESULTS FOR THIS PROCESS

4/25/2012 6:28:25 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Sorry I do not have gel pictures of E coli genomic DNA at this moment--in my lab notebooks at my previous lab (for my Ph.D. study).

8/17/2013 11:42:28 AM  

gugu ndiema
ukzn westvile

hey......can I ask something?

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2/1/2012 12:06:54 AM  

sometimes the DNA bands wont be properly come out in agarose gel. can u please tell me why and suggetion to do it properly .please tell me the discussion elobratelly n suggest an articles to read about this in detail

2/3/2012 1:52:37 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

It would be helpful if you could be more specific about “DNA bands wont be properly come out in agarose gel”. For, E.coli genomic DNA, you expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW) on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded. Two possible reasons that may cause DNA degradation in this protocol: make sure do not vigorously vortex and pipet of DNA solutions at step6 or subsequent steps. It may have some DNases contamination in your sample. DNases released from the lysed cells are supposed to be removed by phenol and chloroform extraction (involves protein denaturation too). So, make sure reagents and materials you use are DNase –free ("Molecular Biology" grade reagents), especially RNase. You can try to check your DNA on the gel before you treat it with RNase.

Hope I answered your questions. Another option is to try some commercial kits, like QIAamp DNA Mini Kit. They should be good, but expensive. (I personally nerve tried them though)

2/4/2012 12:39:56 PM  

abirami selvaraj
sri ramachandra university

ya i got it thank you so much

8/31/2013 10:54:34 AM  

Palak Parashar
Delhi University

why we use e.coli for plasmid dna extraction, why not any other bacteria

11/21/2013 11:57:23 PM  

mamta raghav
Jaypee University Solan, India

I am doing DNA isolation now a days....when picking aqueous phase white protein layer comes along, actually the white layer is not separate its theard are there in aq. phase also.... why???

4/4/2016 9:26:34 AM  

Anthony Bayega
McGill University

same here, I think it's because this is high molecular weight DNA so you will need to pass the pass the whole lysate 2-5 times through a needle (I use a 27gauge needle and 1ml syringe) then proceed to the first phenol-chloroform extraction

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