Difference between revisions of "Part:BBa K1188007"
 
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<b>Step-by-step Protocol</b>
+
<b>Materials</b>
 +
 
 +
  •  Standard Gel Electrophoresis Equipment
 +
  •  Digested DNA Sample (preferably @ 20ng/μL)
 +
  •  1x - 1.5 mL Eppendorf Tube
 +
  •  1x - 0.65 mL Micro-centrifuge Tube
 +
  •  Syringe
 +
  •  Razor Blade
 +
  •  Tweezers (optional)
 +
  •  Filter Paper
 +
  •  Dialysis Tubing
 +
 
 +
<b>Step-By-Step Protocol</b>
  
 
   1.  Prepare a standard agarose gel
 
   1.  Prepare a standard agarose gel
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           - 1.0% = ~0.75g - small fragments (<1000 bp)
 
           - 1.0% = ~0.75g - small fragments (<1000 bp)
 
           - 0.7% = ~0.525g - large fragments (~1000+ bp)
 
           - 0.7% = ~0.525g - large fragments (~1000+ bp)
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           - 7.5μL SYBR®Safe stain
+
           - 7.5μL - 1000x SYBR®Safe stain
 
   2.  Load DNA w/ loading dye into the gel
 
   2.  Load DNA w/ loading dye into the gel
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       o  Load 25μL digest (500 ng) w/ 5μL of 6x loading dye
+
       o  Load 25μL digest (500 ng) w/ 5μL of 6x loading dye (30μL total)
 
       o  Leave at least one lane between samples
 
       o  Leave at least one lane between samples
 
   3.  Run gel for ~30-40 min. @ 120 V until ladder and bands are fully resolved
 
   3.  Run gel for ~30-40 min. @ 120 V until ladder and bands are fully resolved
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   8.  Remove the filter paper and dialysis tubing from the gel and place them in the micro-centrifuge tube
 
   8.  Remove the filter paper and dialysis tubing from the gel and place them in the micro-centrifuge tube
 
   9.  Spin for 30 s. @ 13,200 RPM to collect the purified sample in the 1.5 mL Eppendorf tube
 
   9.  Spin for 30 s. @ 13,200 RPM to collect the purified sample in the 1.5 mL Eppendorf tube
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   10. Remove the micro-centrifuge tube and proceed to ligation or store @ -20° C
+
   10. Remove the micro-centrifuge tube and proceed to ligation or store @ -20° C
  
  
 
<b>GoPro Video</b>
 
<b>GoPro Video</b>
 +
  
 
<html>
 
<html>
 
<video width="320" height="240" controls>
 
<video width="320" height="240" controls>
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<source src="https://static.igem.org/mediawiki/2013/b/be/GelExtraction.mp4" type="video/mp4">
 
 
<source src="https://static.igem.org/mediawiki/2013/1/11/GelExtraction.webm" type="video/webm">
 
<source src="https://static.igem.org/mediawiki/2013/1/11/GelExtraction.webm" type="video/webm">
 +
<source src="https://static.igem.org/mediawiki/2013/b/be/GelExtraction.mp4" type="video/mp4">
 
<a href="https://static.igem.org/mediawiki/2013/b/be/GelExtraction.mp4">GoPro Protocol: Agarose Protein Separation (MP4)</a>
 
<a href="https://static.igem.org/mediawiki/2013/b/be/GelExtraction.mp4">GoPro Protocol: Agarose Protein Separation (MP4)</a>
 
<a href="https://static.igem.org/mediawiki/2013/1/11/GelExtraction.webm">GoPro Protocol: Agarose Protein Separation (WebM)</a>
 
<a href="https://static.igem.org/mediawiki/2013/1/11/GelExtraction.webm">GoPro Protocol: Agarose Protein Separation (WebM)</a>
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<b>Protocol - Tabulated for Quick Reference</b>
 
<b>Protocol - Tabulated for Quick Reference</b>
 +
  
 
<html>
 
<html>
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<b>Additional comments:</b>
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<b>Additional Comments:</b>
  
 
   •  The micro-centrifuge tube serves as a filter and the Eppendorf tube as a collection tube for the extracted  
 
   •  The micro-centrifuge tube serves as a filter and the Eppendorf tube as a collection tube for the extracted  
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   •  The purpose of the filter paper is to absorb the DNA and the dialysis tubing is used to impede migration  
 
   •  The purpose of the filter paper is to absorb the DNA and the dialysis tubing is used to impede migration  
 
     through the gel
 
     through the gel
−
   •  Sizing filter paper and dialysis tubing too small makes it difficult to capture all of the sample, too large  
+
   •  Sizing filter paper and dialysis tubing - too small makes it difficult to capture all of the sample, too large  
 
     results in a more dilute sample
 
     results in a more dilute sample
 
   •  Make sure to have a sharp razor blade or scalpel, it is ok to cut all the way across the gel if desired
 
   •  Make sure to have a sharp razor blade or scalpel, it is ok to cut all the way across the gel if desired
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   •  After removing the filter paper and dialysis tubing, imaging of the gel should show that the band has been  
 
   •  After removing the filter paper and dialysis tubing, imaging of the gel should show that the band has been  
 
     completely removed from the gel
 
     completely removed from the gel
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<!-- -->
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K1188007 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display
 
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===Functional Parameters===
 
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<partinfo>BBa_K1188007 parameters</partinfo>
 
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<!-- -->
 

Latest revision as of 03:03, 21 October 2013

Gel Extraction of DNA


Materials 

 •  Standard Gel Electrophoresis Equipment
 •  Digested DNA Sample (preferably @ 20ng/μL)
 •  1x - 1.5 mL Eppendorf Tube
 •  1x - 0.65 mL Micro-centrifuge Tube
 •  Syringe
 •  Razor Blade
 •  Tweezers (optional)
 •  Filter Paper
 •  Dialysis Tubing

Step-By-Step Protocol 

 1.  Prepare a standard agarose gel
     o  0.7-1.0% agarose to ~75mL of 1x TAE
          - 1.0% = ~0.75g - small fragments (<1000 bp)
          - 0.7% = ~0.525g - large fragments (~1000+ bp)
          - 7.5μL - 1000x SYBR®Safe stain
 2.  Load DNA w/ loading dye into the gel
     o  Load 25μL digest (500 ng) w/ 5μL of 6x loading dye (30μL total)
     o  Leave at least one lane between samples
 3.  Run gel for ~30-40 min. @ 120 V until ladder and bands are fully resolved
     o  Prepare materials for extraction
          - Puncture a hole in the bottom of a 0.65 mL micro-centrifuge tube with a syringe
          - Place the micro-centrifuge tube inside a 1.5 mL Eppendorf tube
          - Cut filter paper and dialysis tubing into small rectangles such that they are slightly wider than the 
            loading well and slightly taller than the height of the gel
 4.  Use a razor blade or scalpel to make an incision just downstream of the band for the segment you wish to 
     purify
 5.  Insert the filter paper and dialysis tubing such that the filter paper is between the DNA sample and the 
     dialysis tubing
 6.  Run the gel for ~ 5 min. @ 120 V
 7.  Image the gel to ensure that DNA is trapped within the filter paper and dialysis tubing
 8.  Remove the filter paper and dialysis tubing from the gel and place them in the micro-centrifuge tube
 9.  Spin for 30 s. @ 13,200 RPM to collect the purified sample in the 1.5 mL Eppendorf tube
 10. Remove the micro-centrifuge tube and proceed to ligation or store @ -20° C


GoPro Video



Protocol - Tabulated for Quick Reference


Gel Extraction Protocol


Additional Comments: 

 •  The micro-centrifuge tube serves as a filter and the Eppendorf tube as a collection tube for the extracted 
    sample
 •  The purpose of the filter paper is to absorb the DNA and the dialysis tubing is used to impede migration 
    through the gel
 •  Sizing filter paper and dialysis tubing - too small makes it difficult to capture all of the sample, too large 
    results in a more dilute sample
 •  Make sure to have a sharp razor blade or scalpel, it is ok to cut all the way across the gel if desired
 •  Place the gel back into buffer slowly so that it does not wash the filter paper and dialysis tubing out of the 
    gel
 •  After removing the filter paper and dialysis tubing, imaging of the gel should show that the band has been 
    completely removed from the gel