Corum:Gel Purification: Difference between revisions
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# Add 3V gel solubilization buffer. Incubate 55 °C 10 min. | # Add 3V gel solubilization buffer. Incubate 55 °C 10 min. | ||
# Vortex to ensure gel is completely dissolved. If it is not, continue incubating 55 °C and vortexing until complete dissolution. | # Vortex to ensure gel is completely dissolved. If it is not, continue incubating 55 °C and vortexing until complete dissolution. | ||
# Incubate RT 2 min. | |||
# Add 1V isopropynol. Vortex and spin. | # Add 1V isopropynol. Vortex and spin. | ||
# Apply to PureLink gel extraction column. Spin in tabletop centrifuge | # Apply to PureLink gel extraction column. Spin in tabletop centrifuge max speed 1 min. Discard flowthrough. | ||
# Apply | # Apply 500 μL wash buffer. Spin max speed 1 min. Discard flowthrough. | ||
# Spin max speed 2 min to dry. | # Spin max speed 2 min to dry. | ||
# Apply 100 μL sterile ddH<sub>2</sub>O to column membrane. Take care to not touch membrane with pipet tip. | # Apply 100 μL sterile ddH<sub>2</sub>O to column membrane. Take care to not touch membrane with pipet tip. |
Revision as of 16:54, 19 June 2012
- SC 16:00, 19 June 2012 (EDT):
Overview
Gel Purification using Invitrogen's PureLink Quick Gel Extraction Kit.
Materials
- Gel embedded DNA
- PureLink gel extraction column
- Gel solubilization buffer (L3)
- Isopropynol
- Wash buffer (W1)
- Sterile ddH2O
Procedure
- Extract DNA bearing piece of the gel using a razor blade.
- Weight the gel piece in mg. This number is defined as V.
- Add 3V gel solubilization buffer. Incubate 55 °C 10 min.
- Vortex to ensure gel is completely dissolved. If it is not, continue incubating 55 °C and vortexing until complete dissolution.
- Incubate RT 2 min.
- Add 1V isopropynol. Vortex and spin.
- Apply to PureLink gel extraction column. Spin in tabletop centrifuge max speed 1 min. Discard flowthrough.
- Apply 500 μL wash buffer. Spin max speed 1 min. Discard flowthrough.
- Spin max speed 2 min to dry.
- Apply 100 μL sterile ddH2O to column membrane. Take care to not touch membrane with pipet tip.
- Incubate RT 10 min.
- Spin max speed 2 min to elute.
- Optional: Combine like samples and/or vacuum centrifuge at 60 °C to reduce volume. IMPORTANT: Time depends on initial and desired final volumes; samples should be checked frequently to prevent total evaporation.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers, books, and websites
Contact
or instead, discuss this protocol.