Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up
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(New page: {{:Team:LMU-Munich/Templates/Page Header}} <b>Gel extraction or PCR Clean Up</b> Source:http://www.biodee.net/UploadFile/Up-2009-11-26_633948249752287808-a9281.pdf <b>Gel Slice and PCR Pr...) |
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{{:Team:LMU-Munich/Templates/Page Header}} | {{:Team:LMU-Munich/Templates/Page Header}} | ||
<b>Gel extraction or PCR Clean Up</b> | <b>Gel extraction or PCR Clean Up</b> | ||
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Source:http://www.biodee.net/UploadFile/Up-2009-11-26_633948249752287808-a9281.pdf | Source:http://www.biodee.net/UploadFile/Up-2009-11-26_633948249752287808-a9281.pdf | ||
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+ | Note: from 10ng to 40µg of DNA can be bound by the membrane (don't try to eluate more, as membrane could be blocked) | ||
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<b>Gel Slice and PCR Product Preparation</b> | <b>Gel Slice and PCR Product Preparation</b> | ||
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A. Dissolving the Gel Slice | A. Dissolving the Gel Slice | ||
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1. Following electrophoresis, excise DNA band from gel and place gel slice in a | 1. Following electrophoresis, excise DNA band from gel and place gel slice in a | ||
1.5ml microcentrifuge tube. | 1.5ml microcentrifuge tube. | ||
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2. Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and | 2. Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and | ||
- | incubate at 50–65°C until gel slice is completely dissolved. | + | incubate at 50–65°C (we used 95°C as the agar didn't melt at 60°C) until gel slice is completely dissolved. |
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B. Processing PCR Amplifications | B. Processing PCR Amplifications | ||
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1. Add an equal volume of Membrane Binding Solution to the PCR amplification. | 1. Add an equal volume of Membrane Binding Solution to the PCR amplification. | ||
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<b>Binding of DNA</b> | <b>Binding of DNA</b> | ||
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1. Insert SV Minicolumn into Collection Tube. | 1. Insert SV Minicolumn into Collection Tube. | ||
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2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn | 2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn | ||
assembly. Incubate at room temperature for 1 minute. | assembly. Incubate at room temperature for 1 minute. | ||
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3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert | 3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert | ||
Minicolumn into Collection Tube. | Minicolumn into Collection Tube. | ||
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<b>Washing</b> | <b>Washing</b> | ||
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4. Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g | 4. Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g | ||
for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection | for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection | ||
Tube. | Tube. | ||
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5. Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g | 5. Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g | ||
for 5 minutes. | for 5 minutes. | ||
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6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute | 6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute | ||
with the microcentrifuge lid open (or off) to allow evaporation of any residual | with the microcentrifuge lid open (or off) to allow evaporation of any residual | ||
ethanol. | ethanol. | ||
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<b>Elution</b> | <b>Elution</b> | ||
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7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. | 7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. | ||
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8. Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room | 8. Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room | ||
temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. | temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. | ||
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9. Discard Minicolumn and store DNA at 4°C or –20°C. | 9. Discard Minicolumn and store DNA at 4°C or –20°C. | ||
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{{:Team:LMU-Munich/Templates/Page Footer}} | {{:Team:LMU-Munich/Templates/Page Footer}} |
Latest revision as of 13:40, 19 August 2010
Source:http://www.biodee.net/UploadFile/Up-2009-11-26_633948249752287808-a9281.pdf
Note: from 10ng to 40µg of DNA can be bound by the membrane (don't try to eluate more, as membrane could be blocked)
A. Dissolving the Gel Slice
1. Following electrophoresis, excise DNA band from gel and place gel slice in a
1.5ml microcentrifuge tube.
2. Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and
incubate at 50–65°C (we used 95°C as the agar didn't melt at 60°C) until gel slice is completely dissolved.
1. Add an equal volume of Membrane Binding Solution to the PCR amplification.
1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn
assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert
Minicolumn into Collection Tube.
4. Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g
for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection
Tube.
5. Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g
for 5 minutes.
6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute
with the microcentrifuge lid open (or off) to allow evaporation of any residual
ethanol.
7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
8. Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room
temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
9. Discard Minicolumn and store DNA at 4°C or –20°C.
Gel extraction or PCR Clean Up
Gel Slice and PCR Product Preparation
B. Processing PCR Amplifications
Binding of DNA
Washing
Elution