Team:UNICAMP-EMSE Brazil/protocols/DNA gel extraction

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==<font size="5" color=#086A87>DNA gel extraction:==
==<font size="5" color=#086A87>DNA gel extraction:==
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'''Purification of gel bands''' ( Fermentas GENEJET gel extraction kit – adapted)
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Extract the DNA from the band with appropriate kit as manufacturer’s instructions.
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:'''1.''' Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.
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:'''2.''' Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)
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:'''3.''' Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
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:'''4.''' Transfer up to 800 μl of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
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:'''5.''' Add 700 μl of Wash Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
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:'''6.''' Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
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:'''7.''' Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube. Add 50 μl of  pre-heated (60˚C) sterile water to the center of the purification column membrane. Centrifuge for 1 min
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:'''8.''' Store the purified DNA at -20˚C.
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Latest revision as of 15:42, 27 September 2011

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UNICAMP-EMSE Brazil Cut dna gel.png

DNA gel extraction:

Purification of gel bands ( Fermentas GENEJET gel extraction kit – adapted)

1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.
2. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)
3. Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
4. Transfer up to 800 μl of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
5. Add 700 μl of Wash Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
6. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
7. Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube. Add 50 μl of pre-heated (60˚C) sterile water to the center of the purification column membrane. Centrifuge for 1 min
8. Store the purified DNA at -20˚C.













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