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Extracting Large DNA Plasmids from Agarose Gels

Extracting Large DNA Plasmids from Agarose Gels - Molecular Biology Techniques

Extracting Large DNA Plasmids from Agarose Gels - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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  #1  
Old 07-04-2006, 08:30 AM
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Default Extracting Large DNA Plasmids from Agarose Gels



Dear All members
i want to extract plasmids from agarose gel, its
size is about 15KB, i have tried commercial kit for
DNA purification by QAGIN and INVITROGEN, but could
not succeed, kindly guide me and give some other
protocol for it
thanks in advance
najma
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  #2  
Old 07-04-2006, 04:32 PM
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Default Re: techniques in molecular biology

Dear Najmaarshad,
i had the same problem many times!

Tips for extracting DNA from gels:

1) you need a lot of DNA, preferrably over 20 ugs.

2) you need to heat the gel very well (I use 75-80 C with Qiagen gel extraction kit and mix very well )

3) you can pool several agarose gel fragment purifications into ONE column to get it more concentrated

just some tips from me that helped me tremendously

actually your plasmid size is 15kb... the Qiagen system doesnt work well with plasmids over 10 kb in size. So I will check to see if there are any protocols available for your size plasmid.

Update:

Ok try the roche kit with the tips I mentioned it seems that extraction works well even with 23 kb plasmids.

[Only registered users see links. ]

Last edited by westsd; 07-04-2006 at 04:38 PM.
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Old 07-04-2006, 04:50 PM
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Arrow Re: Extracting Large DNA Plasmids from Agarose Gels

Hey najmaarshad!

welcome to the forum.
  • Plasmid DNA fragments which are larger than 1 kb tend to become trapped in the agarose matrix if they are not immediately eluted from the agarose.
Use Electro Elution to Extract Large Plasmids from Agarose
  1. Elute plasmid DNA FRESH from agarose, do NOT store the agarose gel slices in the refrigerator as even 1 hour at 4 degrees greatly compounds the problem.
  2. Use some clamps and dialysis tubing. Clamp the bottom of a small dialysis tubing piece and fill it with 0.5 X TBE.
  3. Use Dialysis Tubing with a MW Cut off of 3500.
  4. Use a clean scalpel blade, cut out the gel piece from the gel right after running it.
  5. You can then place the gel into the open, buffer filled dialysis bag.
  6. Squeeze out as much TBE buffer as you can without introducing air bubbles.
  7. Clamp the top of the dialysis tubing.
  8. Place the clamped dialysis tubing horizontal in an agarose gel box.
  9. Run at 15 watts for 50-90 minutes.
  10. Reverse the polarity after running for 90 seconds to direct the DNA back off of the membrane.
  11. The gel slice should be as small as possible and DNA should be eluted through the thinnest dimension.
  • If you are electro-eluting ethidium bromide-stained DNA, you can very briefly expose the dialysis bag containing the gel slice on the UV transiluminator to see if DNA remains in thet gel slice.
  • You can repeat elution if required in a fresh piece of tubing.
  • Ethanol precipitate the DNA if you want to concentrate it.
  • You can then run a small amount on a gel to visualize the bands stained with Ethidium bromide or SYBR DNA dye.
The previous was based on the method from :
http://www.ncbi.nlm.nih.gov/entrez/q...&dopt=Abstract
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Old 07-04-2006, 04:52 PM
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Default Re: Extracting Large DNA Plasmids from Agarose Gels

More protocols for DNA electro elution from agarose gels:

http://rothlab.ucdavis.edu/protocols...elution-2.html

http://humgen.wustl.edu/hdk_lab_manual/rdm/rdm9.html

http://rothlab.ucdavis.edu/protocols...roelution.html
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Old 07-04-2006, 04:53 PM
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Default Re: Extracting Large DNA Plasmids from Agarose Gels

Special Apparatus for DNA electro elution:

http://www.gerardbiotech.com/electoelution.htm
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Old 07-05-2006, 08:55 AM
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Default Re: Extracting Large DNA Plasmids from Agarose Gels

Respected Sir/Madam
thanks a lot for the guidence, i shall try to extract plasmid by following your suggessions.
regards
najma
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Old 08-24-2006, 06:19 PM
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Default Re: Extracting Large DNA Plasmids from Agarose Gels

Hey Najmaarshad,
please tell me how it worked out... I am going to try it soon as well.

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