Difference between revisions of "Part:BBa K1188007"

Revision as of 19:39, 12 October 2013

Gel Extraction of DNA

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 SYBR®Safe stain
 2.  Load DNA w/ loading dye into the gel
     o  Load 25μL digest (500 ng) w/ 5μL of 6x loading dye
     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

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

Sequence and Features