User:Norman Wang/Agarose Gel Liquid-Band Extraction

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  • One step extraction of DNA from agarose gel electrophoresis without Gel Cutting & Purification.

Contents

Methods

Materials

  • 1.2% Agarose Gel with SYBR Safe (glows in both Blue Light and UV)
  • UPA + 2 Log Ladder Mixture
  • use Blue light 470nm LED illumination while extracting band (so it does not damage DNA)
  • use UV illumination (more clearly visible) post extraction to verify

Protocol

  1. Cast gel using wide combs (x6 lanes), and line up the two sets of combs. First set comb wells for loading DNA, second set comb wells for extraction.
  2. Load enough buffer to slightly submerge agarose gel, leave thin layer of top surface un-submerged (so DNA doesn't run out of second row of wells)
  3. Prefill first set of wells with Nanopure H2O or buffer (if it has not already been accidentally flooded)
  4. Prefill second set of wells with Nanopure H2O
  5. Run until desired band gets close to second set of wells, use blue light so we don't damage DNA.
  6. Closely monitor band moving into extraction wells (second row of wells), use camera long shutter photograph to verify if band is too faint to see. Use flashlight to assist locating the well (flash on/off to see extraction well location/band fluorescence).
  7. Stop electrophoresis when band migrates into H2O filled well.
  8. Pickup DNA from second well using pipetter.
  9. After complete pickup, continue running the gel so band (if not picked up) continues to migrate.
  10. Later verify via photo (under UV or Blue Light illumination) that "some" if not all of the DNA band has been picked-up.
  11. Concentrate DNA and clean up salts from electrophoresis using Ethanol_precipitation_of_nucleic_acids

Results

  • DNA was successfully picked up via this method, yield is low. Need more practice.
  • Would help if I load more DNA next time!
Extraction Verified
Extraction Verified
Extraction in progress, tracking the migrating 408bp band
Extraction in progress, tracking the migrating 408bp band

Discussion

Future Improvements

  • Load more than just 10uL of DNA (from PCR or Restriction Digest)
  • Cast gel with:
    1. Thin loading (upper) well comb (for clearly defined running band)
    2. Fat pickup (lower) well comb (so it does not run back into gel immediately)
  • Need brighter blue light illumination?
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