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

Materials

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

• 

  thin filter, easy to insert, less volume, bigger chance DNA may elude the trap

• 

  thick filter, harder to insert, bigger volume, smaller chance DNA may elude the trap

• Dialysis Tubing 3500mwco

Simplified Procedure (original detailed procedure [1])

Gel Band Extraction:

1. run a gel... make sure that the band you want to cut is clearly separated from the rest.
2. use a blue light box to see SYBR Safe stained bands; for EtBR stained DNA, use long-wavelength UV to avoid DNA damage.
3. using a razor, make an incision below the band, slightly wider than the band itself.
4. insert filter/membrane sandwich into the incision. Place glass filter closer to band and dialysis membrane away from band into incision in one smooth action.
5. place gel tray back into box and run for 5 minutes, ensure the band runs into the filter/membrane sandwich.
6. take filter/membrane sandwich out of gel, and shove into a spin column (a smaller tube with hole on bottom, or a qiaquick spin column without its silica membrane) within a collection eppendorf tube. Place filter/membrane sandwich with filter facing down and membrane on top.
7. centrifuge for 1 minutes at 13k rpm
8. take elute and purify using QIAquick PCR Purification Protocol.

QIAquick PCR Purification Protocol:

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.

   For example, add 500 μl of Buffer PB to 100 μl PCR sample.

2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
4. Discard flow-through. Place the QIAquick column back into the same tube.

   Collection tubes are re-used to reduce plastic waste.

5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
6. Discard flow-through and place the QIAquick column back in the same tube.

   Centrifuge the column for an additional 1 min.

   IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

7. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. dry in vacuum for 5 minutes, ensure all ethanol has evaporated
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

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 Safe using recommended concentration.

• 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 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

‡ The gel experiments above were performed by Megan Nakashima and Norman Wang

References

1. Original detailed procedure with precautions & notes. Personal communication with David Thompson of Liu Lab @ Harvard