Team:RHIT/Protocols/GelExtraction

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Notes before starting:

• This protocol is for the purification of up to 10 µg DNA.
• The yellow color of Buffer QG indicates a pH ≤ 7.5. DNA absorption to the membrane is only efficient at pH ≤ 7.5.
• Add ethanol (96%-100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%) and a heating block or water bath at 50°C are required.
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

Procedure

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100µL). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.


3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to the Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.


4. Add 1 volume isopropanol to the sample and mix.


5. Place a QIAquick spin column in the microcentrifuge in a provided 2 mL collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µL, load and spin again.


6. If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 µL Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick spin column back into the same tube.



7. To wash, add 750 &#181L Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.

   
   

   NOTE: If the DNA will be used for salt-sensitive applications (e.g. sequencing, blunt-ended ligation), let the column stand for 2-5 min after addition of Buffer PE.

   
   

   Centrifuge the QIAquick column in the provided 2 mL collection tube for 1 min to remove residual wash buffer.



8. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.


9. To elute DNA, add 50 µL Buffer EB (10 mM Tris Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of the Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.


10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.