Difference between revisions of "Elute the released products"

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# Add 0.75 ml of Buffer PE to QIAquick column, let it stand for 2 to 5 minutes and then centrifuge for 1 min at 13,000 rpm.
 
# Add 0.75 ml of Buffer PE to QIAquick column, let it stand for 2 to 5 minutes and then centrifuge for 1 min at 13,000 rpm.
 
#Discard the flow-through and centrifuge the QIAquick column for another minute at 13,000 rpm.
 
#Discard the flow-through and centrifuge the QIAquick column for another minute at 13,000 rpm.
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#The QIAquick column which is a small disc of silica gel should then be placed in a fresh eppendorff tube.
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#The QIAquick column should then be placed in a fresh eppendorff tube.
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#To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the
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#To elute (extract) the DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane (a small disc of silica gel) and centrifuge the column for 1 min.<br/> For stronger concentration of DNA, add 30 μl elution buffer to the center of the QIAquick membrane,
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QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased
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let it stand for 1 minute, and then centrifuge for 1 minute.
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DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane,
 
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let the column stand for 1 min, and then centrifuge for 1 min.
 
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IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick
 
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membrane for complete elution of bound DNA. The average eluate volume is 48 μl
 
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from 50 μl elution buffer volume, and 28 μl from 30 μl.
 
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Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
 
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between pH 7.0 and 8.5. When using water, make sure that the pH value is within
 
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this range, and store DNA at –20°C as DNA may degrade in the absence of a
 
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buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM
 
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EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
 

Revision as of 15:50, 15 June 2009

The Procedure

  1. Remove the DNA fragments from the agarose gel with a clean scalpel. Try to get as small a piece of gel as possible, without touching the DNA.
  2. Weigh the gel slice in a clorless tube. Add 3 times the volume of Buffer QG to 1 volume of gel.
  3. Incubate at 50°C for 10 minutes, or until all the gel has dissolved. Mix the tube using the vortex mixer every 2 or 3 minutes to make sure it dissolves.
  4. Check if the mixture is yellow in color. If it is, it means that its pH is around 7.5. This is the pH at which the adsorbtion (stickiness)of the QIAquick membrane is most efficient. If it is orange or violet, it is too acidic. Add 10µl of 3 M sodium acetate (pH 5.0) and mix, if that is the case.
  5. Add isopropanol of a volume equal to the volume of the gel to the sample and mix. The isopropanol precipitates the DNA.(?)
  6. Place QIAquick spin column in a 2ml collection tube.
  7. To collect the DNA, pour the sample into the QIAquick column, and centrifuge for 1 min.
  8. Discard flow-through and place QIAquick column back in the same collection tube.
    Collection tubes are re-used to reduce plastic waste.
  9. Add 0.75 ml of Buffer PE to QIAquick column, let it stand for 2 to 5 minutes and then centrifuge for 1 min at 13,000 rpm.
  10. Discard the flow-through and centrifuge the QIAquick column for another minute at 13,000 rpm.
  11. The QIAquick column should then be placed in a fresh eppendorff tube.
  12. To elute (extract) the DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane (a small disc of silica gel) and centrifuge the column for 1 min.
    For stronger concentration of DNA, add 30 μl elution buffer to the center of the QIAquick membrane,

let it stand for 1 minute, and then centrifuge for 1 minute.

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