Valencia/Purification
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#*Electrophorese until DNA band of interest is isolated from adjacent contaminating fragments | #*Electrophorese until DNA band of interest is isolated from adjacent contaminating fragments | ||
#Identify bands by staining gel with ethidium bromide or SYBR Green I or Nucleic Acid Gel Stain. | #Identify bands by staining gel with ethidium bromide or SYBR Green I or Nucleic Acid Gel Stain. | ||
- | #*[[Image: | + | #*[[Image:VAdv.jpg ]]Wear gloves, ethidium bromide is a known potent carcinogen. |
#Cut desired DNA band from gel using an ethanol-cleaned scalpel or razor blade. | #Cut desired DNA band from gel using an ethanol-cleaned scalpel or razor blade. | ||
- | #*[[Image: | + | #*[[Image:VAdv.jpg ]]Minimize gel volume by visualizing DNA and cutting the smallest possible gel slice on a UV light box. |
#Place excised agarose gel slice in a sterile 1.5 ml microcentrifuge tube. | #Place excised agarose gel slice in a sterile 1.5 ml microcentrifuge tube. | ||
#*Determine gel mass by first pre-weighting the tube, and then reweighting the tube with the excised gel slice. | #*Determine gel mass by first pre-weighting the tube, and then reweighting the tube with the excised gel slice. | ||
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#Insert one High Pure Filter Tube into one Collection Tube. | #Insert one High Pure Filter Tube into one Collection Tube. | ||
#*Pipette the entire contents of the microcentrifuge tube into the upper reservoir of the Filter Tube. | #*Pipette the entire contents of the microcentrifuge tube into the upper reservoir of the Filter Tube. | ||
- | #*[[Image: | + | #*[[Image:VAdv.jpg ]]Do not exceed 700 μl total volume. If mixture is > 700 μl, split the volume and use two separate Filter Tubes for each portion. |
#Centrifuge 30 - 60 s at maximum speed in a standard table top centrifuge at +15 to +25°C. | #Centrifuge 30 - 60 s at maximum speed in a standard table top centrifuge at +15 to +25°C. | ||
#*Discard the flowthrough solution. | #*Discard the flowthrough solution. | ||
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#*Add 200 μl Wash Buffer. | #*Add 200 μl Wash Buffer. | ||
#*Centrifuge 1 min at maximum speed. | #*Centrifuge 1 min at maximum speed. | ||
- | #*[[Image: | + | #*[[Image:VLupa.jpg ]]This second 200 μl wash step ensures optimal purity and completeremoval of Wash Buffer from the glass fibers. |
#Discard the flowthrough solution and Collection Tube. | #Discard the flowthrough solution and Collection Tube. | ||
#*Recombine Filter Tube with a clean 1.5 ml microcentrifuge tube. | #*Recombine Filter Tube with a clean 1.5 ml microcentrifuge tube. | ||
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#*Centrifuge 1 min at maximum speed. | #*Centrifuge 1 min at maximum speed. | ||
#The microcentrifuge tube now contains the purified DNA. | #The microcentrifuge tube now contains the purified DNA. | ||
- | #*[[Image: | + | #*[[Image:VAdv.jpg ]]When subsequent OD260 determination is planned, centrifuge the eluate for more than 1 min at maximum speed to remove residual glass fibers from the eluate, because they may disturb absorbance measurement. Use an aliquot of the supernatant to determine concentration. |
- | #*[[Image: | + | #*[[Image:VLupa.jpg ]]Either use the eluted DNA directly or store the eluted DNA at +2 to +8°C or -15 to -25°C for later analysis. |
Latest revision as of 06:47, 26 September 2007
Information obtained from:
Roche Applied Science: High Pure PCR Purification Kit
Protocol obtained from: High Pure PCR Purification Kit
Pdf link: Pdf
The following protocol is for the purification procedure for DNA from a 100 mg agarose gel slice
- Isolate DNA band of interest electrophoretically as follows.
- Load PCR reaction mixture on a 0.8 - 2% agarose gel.
- Use 1 × TAE or 1 × TBE as running buffer.
- Electrophorese until DNA band of interest is isolated from adjacent contaminating fragments
- Identify bands by staining gel with ethidium bromide or SYBR Green I or Nucleic Acid Gel Stain.
- Cut desired DNA band from gel using an ethanol-cleaned scalpel or razor blade.
- Place excised agarose gel slice in a sterile 1.5 ml microcentrifuge tube.
- Determine gel mass by first pre-weighting the tube, and then reweighting the tube with the excised gel slice.
- Add 300 μl Binding Buffer for every 100 mg agarose gel slice to the microcentrifuge tube.
- Dissolve agarose gel slice in order to release the DNA:
- Vortex the microcentrifuge tube 15 - 30 s to resuspend the gel slice in the Binding Buffer.
- Incubate the suspension for 10 min at 56°C.
- Vortex the tube briefly every 2 - 3 min during incubation.
- After the agarose gel slice is completely dissolved:
- Add 150 μl isopropanol for every 100 mg agarose gel slice to the tube.
- Vortex thoroughly.
- Insert one High Pure Filter Tube into one Collection Tube.
- Centrifuge 30 - 60 s at maximum speed in a standard table top centrifuge at +15 to +25°C.
- Discard the flowthrough solution.
- Reconnect Filter Tube with the same Collection Tube.
- Add 500 μl Wash Buffer to the upper reservoir.
- Centrifuge 1 min at maximum speed (as above).
- Discard the flowthrough solution.
- Discard the flowthrough solution and Collection Tube.
- Recombine Filter Tube with a clean 1.5 ml microcentrifuge tube.
- Add 50 - 100 �l Elution Buffer to the upper reservoir of the Filter Tube.
- Centrifuge 1 min at maximum speed.
- The microcentrifuge tube now contains the purified DNA.
- When subsequent OD260 determination is planned, centrifuge the eluate for more than 1 min at maximum speed to remove residual glass fibers from the eluate, because they may disturb absorbance measurement. Use an aliquot of the supernatant to determine concentration.
- Either use the eluted DNA directly or store the eluted DNA at +2 to +8°C or -15 to -25°C for later analysis.