Corum:Gel Purification: Difference between revisions
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==Materials== | ==Materials== | ||
* DNA embedded in gel. | * DNA embedded in gel | ||
* Purelink column | |||
* Gel solubilization buffer | |||
* Isopropynol | |||
* Wash buffer | |||
* Elution buffer or sterile ddH<sub>2</sub>O. | |||
==Procedure== | ==Procedure== | ||
| Line 17: | Line 22: | ||
# Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough. | # Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough. | ||
# To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube. | # To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube. | ||
# Apply 50-100 μL elution buffer or | # Apply 50-100 μL elution buffer or sterile ddH<sub>2</sub>O to column membrane. Avoid touching membrane with tip. Incubate RT 10 min. | ||
# To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube. | # To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube. | ||
# Quantify DNA sample by [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore]. | # Quantify DNA sample by [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore]. | ||
Latest revision as of 15:00, 25 July 2012
Overview
Gel extraction with Invitrogen Purelink Gel Extraction Kit.
Materials
- DNA embedded in gel
- Purelink column
- Gel solubilization buffer
- Isopropynol
- Wash buffer
- Elution buffer or sterile ddH2O.
Procedure
- Excise DNA from gel with razor blade. Place in 1.5 mL tube and weigh gel piece. Let V be the weight of the piece in mg.
- Add 3V μL Gel Solubilization Buffer. Incubate 55 °C 10 min or until gel is completely dissolved.
- After gel is completely dissolved, incubate 55 °C additional 5 min.
- Incubate RT 2 min.
- Add 1V μL isopropynol. Mix and spin.
- Apply to column. Centrifuge max speed 1 min. Discard flowthrough. (Maximum application volume is 700 μL, so for larger volumes, repeat until all of the DNA is bound to column membrane.)
- Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough.
- To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube.
- Apply 50-100 μL elution buffer or sterile ddH2O to column membrane. Avoid touching membrane with tip. Incubate RT 10 min.
- To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube.
- Quantify DNA sample by quantifluore.
- Label side of tube with the following:
- [DNA] in nM
- "gel purified"
- date
- Store at -20 °C.
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
Invitrogen Purelink Gel Extraction Kit manual.
Contact
or instead, discuss this protocol.
Digital Signature
- SC 17:57, 25 July 2012 (EDT):
