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Team:EPF-Lausanne/Protocols/Gel purification
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'''Purpose''': separate and extract products of a PCR. | '''Purpose''': separate and extract products of a PCR. | ||
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'''Warning''': the PCR products destined to extraction must be exposed to minimal amounts of UV radiation, to avoid any damage to DNA. | '''Warning''': the PCR products destined to extraction must be exposed to minimal amounts of UV radiation, to avoid any damage to DNA. | ||
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| + | == Equipement == | ||
| + | |||
| + | * '''Gel electrophoresis setup''' | ||
| + | * '''Scalpel''' | ||
| + | * '''Eppendorf tubes''': As many as there are gel samples. The 2 ml ones are necessary if more than 0.3 mg of gel is to be treated. | ||
| + | * '''Qiagen QIAquick gel extraction kit (250)''': make sure the protocol is inside. | ||
| + | * '''UV plate reader''' and UV-shield. | ||
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'''Notes''': | '''Notes''': | ||
* Cut out as little gel as possible, i.e. cut out as close to the band as possible. This will ease the gel extraction. | * Cut out as little gel as possible, i.e. cut out as close to the band as possible. This will ease the gel extraction. | ||
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== DNA Extraction == | == DNA Extraction == | ||
Latest revision as of 08:49, 15 July 2011
Gel purification
Back to protocols.Purpose: separate and extract products of a PCR.
General idea: The PCR products are separated by gel electrophoresis, then mechanically cut out with the gel. The DNA is then extracted from each gel fragment.
Run a first electrophoresis with a low volume of PCR product, to verify the expected fragments were produced. If that is the case, run a new electrophoresis with all the PCR product, using a thicker combs to make larger wells. The well from our thick comb contain up to 25 or 30 ul.
Warning: the PCR products destined to extraction must be exposed to minimal amounts of UV radiation, to avoid any damage to DNA.
Contents |
Equipement
- Gel electrophoresis setup
- Scalpel
- Eppendorf tubes: As many as there are gel samples. The 2 ml ones are necessary if more than 0.3 mg of gel is to be treated.
- Qiagen QIAquick gel extraction kit (250): make sure the protocol is inside.
- UV plate reader and UV-shield.
Cutting the gel
From the first (low-volume) gel, determine which bands contain the PCR products. If necessary, adjust the duration of the second electrophoresis to increase spacing between bands.
- Prepare as many Eppendorf tubes as PCR products to extract. Weigh them.
To cut out a desired band from the gel:
- Lift the black camera box off the UV plate.
- Fix the anti-UV shield on the front of the device; switch on the UV light.
- Quickly identify the desired band, cut around it, and switch off the UV light.
- Remove the cut-out gel and store in an Eppendorf tube.
- Clean the scalpel with DI water before cutting each band to avoid cross-contamination.
Notes:
- Cut out as little gel as possible, i.e. cut out as close to the band as possible. This will ease the gel extraction.
DNA Extraction
Then, to extract the DNA from the gel, use the Qiagen gel purification kit. Follow the included protocol.
Supplementary notes
- Weigh each sample on the microbalance, after taring with an empty tube.
- In the elution step, when you have to centrifuge the column in a new Eppendorf tube, the tube's lid cannot be closed. Leave it open in the centrifuge, oriented towards the bottom right (i.e. behind the tube, as the platter rotates counter-clockwise).
