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Genomic DNA extraction protocol



Genomic DNA extraction protocol

High molecular weight genomic DNA extraction protocol:

Buffer and reagent:
Genomic DNA extraction buffer (250ml):
1M Tris.Cl (pH 8.0) 2.5ml
0.5M EDTA (pH 8.0) 50 ml
Pancreatic RNase 5 mg
10% SDS 12.5 ml
--------------------------------------------------
Adjust pH to 8.0 and adjust volume to 250ml with ddH2O

Saturated phenol (pH 8.0)

10M ammonium acetate (NH4Ac)

Protocol:
1. Weigh 0.5-1g fresh tissue and put in motar. Add liquid nitrogen to
snap freeze tissue and blend tissue to powder.
2. Add 10 ml genomic DNA extraction buffer in 50 ml tube and put
tissue powder in.
3. Invert tube to submerge tissue powder and incubate at 37c for 1 hour.
4. Add 50 ul proteinase K (20mg/ml stock), mix gently.
5. Incubate in 50c water bath for 3 hours, shake gently.
6. Let stand in room temperature for 30 min to equilibrate to room temperature.
7. Add 10 ml Phenol, mix gently for 10 min.
8. Centrifuge at 3000 rpm x 15 min.
9. Transfer the viscous aqueous phase to a new tube using a wide-pore
glass pipette.
10. Repeat phenol extraction for 2 times or more.
11. Add 2 ml ammonium acetate (10M), mix gently.
12. Add 2 volume of ethanol (in room temperature). Swirl gently and
you will see genomic DNA start to form the white mass. Transfer
genomic DNA to a new tube by using a "U" shape pipette.
13. Air dry for 5-10min to drive off ethanol and dissolve in ddH2O or TE buffer.

Reference:
Maniatis et al., Molecular cloning
Protocol for extraction of genomic DNA from swine solid tissues
Promega Notes on Rapid Isolation of High Quality Genomic DNA from Various
Sources (pdf)

Last update 20-Sep-2006, Rating of 11 votes.

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By amir on 12-Mar-2009
Dear SIR,
I am a student of biotec.Ihave tried to extract DNA by non organic method but it not precipitated.Till to protein precipitation no problem but when I treat the supernatent with equql vol.of isopropanol there is no precipitation.I hav also used the 3 M CH3COONa pH 5.2 but no+ive results.
Plz. Sir give me some tips kindly.Ishall be v. thankful to you for this kindness.
Yours truely,
Amir
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By XP on 07-Mar-2009
simpleand good enough, but Centrifuge may use g or not use rpm.
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By vijay on 01-Mar-2009
Dear Sir,

please send to me the protocol to isolating the Plasmid DNA from Agrobacterium culture(BT Gene) .I need such simple protocol as soon as posible I wait your help thankyou very mach .

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By ahmad muhsin on 15-Feb-2009
please send to me the protocol to isolating the DNA from cervical exfoliated cell collected after pap smear .I need such simple protocol as soon as posible I wait your help thankyou very mach .
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By Uma on 13-Feb-2009
Can someone help me with a problem: I am trying to isolate gDNA from mosquitoe larvae but I am unable to get good results(I used ZR genomic isolation kit)
Please reply,
Thanks,
-Uma
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By Absa Jaw on 31-Jan-2009
I was vy pleased to read your work and it was useful to my area of work as a researcher and my current research project as a post graduate in the University of Science and Technology, Ghana..

I would like you to help me with the DNA extraction protocol of rice blast fungus through my email address.I would be very grateful if you can help because I need it very urgent
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By barzanooni on 06-Sep-2008
Dear prof,
I want to extract DNA genomic from halophilic bacteria but I can not find a good protocol. Can you help me?
Thanks.

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By dileep.k on 04-Sep-2008
let us have a still detailed outlook at different protocols supported by the explanation for using perticular reagent/chemical/salt.
thank u.
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By Lai on 02-Sep-2008
Some body have do DNA extraction and purification in bottlenose dolphins?
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By Ossy Shilla on 08-Mar-2007
Please I've found your web very useful especially to my carrer as a researcher and a current prospective postgraduate student in Biotechnology.

Let me have more of these through my email next time in particular, DNA extraction, purification, quantification and analysis. PCR rea\ctions of Simple sequence repeats techniques in form of papers if possible

Thanks
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By egale on 20-Sep-2006
DNA Isolation from Fixed or Paraffin-embedded Tissue Protocol

* Lysis buffer
10mM Tris-Cl (pH8.0)
0.1M EDTA(pH8.0)
0.5%(w/v)SDS
20 ug/ml DNase-free pancreatic RNase
* Xylenes (Fisher cat. no. X5-500)
* Isopropanol (2-propanol)(Fisher cat. no. A464-4)
* 70% Ethanol
* 1.5 ml Sterile microfuge tubes, or 15ml centrifuge tubes
* Micropipettors and pipets
* Vortex mixer
* Microfuge or clinical centrifuge
* Water bath

DNA Isolation from Paraffin-embedded Tissue


Paraffin-embedded Tissue—Sample De-paraffinization

1.Place 50-micron thick scrolls (weight up to 5-10 mg), finely minced, into a 1.5 ml microfuge tube. Add 300 ul Xylene and incubate 5 minutes with constant gentle mixing (for example on a lab rotator) at room temperature.
2.Centrifuge at 13,000-16,000 x g for 1-3 minutes to pellet the tissue. Discard the Xylene.
3.Repeat steps 1- 2, twice (for a total of three Xylene washes).
4.Add 300 ul 100% Ethanol and incubate 5 minutes with constant mixing at room temperature.
5.Centrifuge at 13,000-16,000 x g for 1-3 minutes to pellet the tissue. Discard the ethanol.
6. Repeat steps 4-5 (for a total of two ethanol washes).

Cell Lysis:

1.Add 10 volume deparaffined tissues(V/W) of lysis buffer(aroud 100-300 ul) in sample tube and proteinase K(20mg/ml) to a final lysis buffer, incubate 3 hours at 50C. Swirl tube from time and time.

2.Treatment with phenol
Add equal volume phenol, gently mix 10 minutes and centrifuge 10 minutes at 5000g at RT.Seperate two phases.
3.Use a pipette to transfer the supernanant.(aqueous phases)
repeat extraction with phenol.

4.Transfer aqueous phase to a new tube, add 0.2 volume of 10 M ammonium acetate, and add 2.5 volume 100% ethanol at RT. Mix well.See DNA fragments, centrifuge at 5000g. if not see DNA, freeze tube at -20C over night. centifuge later on.
5. wash with 70% ethanol twice
6. Disslove DNA with 50 ul TE buffer.
7.Store DNA at 4 °C. For long term storage, place sample at -20°C or -80°C.


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