"
Team:EPF-Lausanne/Protocols/Gel purification
From 2011.igem.org
| Line 1: | Line 1: | ||
{{:Team:EPF-Lausanne/Templates/Header|title=Gel purification}} | {{:Team:EPF-Lausanne/Templates/Header|title=Gel purification}} | ||
| - | '''Purpose''': separate and extract products of a PCR | + | '''Purpose''': separate and extract products of a PCR. |
| - | The PCR products are separated by gel electrophoresis, then mechanically cut out with the gel. The DNA is then extracted from each gel fragment. | + | '''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. | 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. | ||
== Cutting the gel == | == 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. | |
| - | * | + | |
| - | * | + | 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. | ||
Revision as of 07:45, 13 July 2011
Gel purification
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.
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.
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.
- To weight your samples, tare the scales with an empty tube
- In the elution step, when you have to centrifuge the column in a new Eppendorf tupe, make sure the lid of the tube flies behind in the centrifugator. It turns counterclockwise.
