User:Norman Wang/DNA Isolation Via Agarose Gel Filter+Membrane Extraction: Difference between revisions

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* Agarose Gel
* Agarose Gel
* DNA Dye: Ethidium Bromide or SYBR Safe (a [http://en.wikipedia.org/wiki/Cyanine_dye Cyanine Dye])
* DNA Dye: Ethidium Bromide or SYBR Safe (a [http://en.wikipedia.org/wiki/Cyanine_dye Cyanine Dye])
* Glass Based Filter [[Image:Whatman_Glass_Fiber_Filter.jpg|thumb|right]][[Image:Fisher_Glass_Fiber_Filter.jpg|thumb|right]]
* Glass Based Filter
* Dialysis Tubing 3500mwco [[Image:Spectra_Por_Dialysis_Tubing_3500MWCO.jpg|thumb|right]]
<gallery>
Image:Whatman_Glass_Fiber_Filter.jpg
Image:Fisher_Glass_Fiber_Filter.jpg
</gallery>
<div style="clear:both">
* Dialysis Tubing 3500mwco
[[Image:Spectra_Por_Dialysis_Tubing_3500MWCO.jpg|thumb|left]]
<div style="clear:both">
== Procedure <cite>Thompson-2008</cite> ==
== Procedure <cite>Thompson-2008</cite> ==
#
 
#
== Yield ==
#
Starting DNA quantity (total in the 10uL loaded)
* 1.0 kb: 124ng
* 1.5 kb: 122ng
* 3.0 kb: 120ng
* 6.0 kb: 48ng
 
Resulting DNA quantity (total in the 10uL eluted)
* 1.0 kb: ng, ng
* 1.5 kb: ng, ng
* 3.0 kb: ng, ng
* 6.0 kb: ng, ng
 
Seven of the eight band extraction yielded some DNA.  We started with no more than 124ng of DNA in each well.  Bands below 1kb were too faint to be visibly stained by SYBR Green using recommended concentration.
 
The experiments were performed by Megan Nakashima and Norman Wang
<gallery caption="Agarose Gel DNA Band Extraction Results">
Image:Filter Membrane Gel Extraction 1kb 1.5kb.annotated.jpg|Extraction of the 1kb and 1.5 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band
Image:Filter Membrane Gel Extraction 3kb 6kb.annotated.jpg|Extraction of the 3kb and 6 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing  extracted band
Image:Filter Membrane Gel Extraction Results.annotated.jpg|Extraction Yield, first row: 1kb and 1.5 kb; second row 3kb and 6kb
</gallery>


== References ==
== References ==

Revision as of 21:34, 15 November 2008

Materials

  • Agarose Gel
  • DNA Dye: Ethidium Bromide or SYBR Safe (a Cyanine Dye)
  • Glass Based Filter
  • Dialysis Tubing 3500mwco

Procedure [1]

Yield

Starting DNA quantity (total in the 10uL loaded)

  • 1.0 kb: 124ng
  • 1.5 kb: 122ng
  • 3.0 kb: 120ng
  • 6.0 kb: 48ng

Resulting DNA quantity (total in the 10uL eluted)

  • 1.0 kb: ng, ng
  • 1.5 kb: ng, ng
  • 3.0 kb: ng, ng
  • 6.0 kb: ng, ng

Seven of the eight band extraction yielded some DNA. We started with no more than 124ng of DNA in each well. Bands below 1kb were too faint to be visibly stained by SYBR Green using recommended concentration.

The experiments were performed by Megan Nakashima and Norman Wang

  • Agarose Gel DNA Band Extraction Results
  • Extraction of the 1kb and 1.5 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band

    Extraction of the 1kb and 1.5 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band

  • Extraction of the 3kb and 6 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band

    Extraction of the 3kb and 6 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band

  • Extraction Yield, first row: 1kb and 1.5 kb; second row 3kb and 6kb

    Extraction Yield, first row: 1kb and 1.5 kb; second row 3kb and 6kb

References

  1. personal communication with David Thompson of Liu Lab @ Harvard

    [Thompson-2008]
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