Corum:Gel Purification: Difference between revisions

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*'''SC 16:00, 19 June 2012 (EDT)''':
==Overview==
==Overview==


Gel Purification using [http://products.invitrogen.com/ivgn/product/K210012 Invitrogen's PureLink Quick Gel Extraction Kit].
Gel extraction with [http://products.invitrogen.com/ivgn/product/K210012 Invitrogen Purelink Gel Extraction Kit].


==Materials==
==Materials==


* Gel embedded DNA
* DNA embedded in gel
* PureLink gel extraction column
* Purelink column
* Gel solubilization buffer (L3)
* Gel solubilization buffer
* Isopropynol
* Isopropynol
* Wash buffer (W1)
* Wash buffer
* Sterile ddH<sub>2</sub>O
* Elution buffer or sterile ddH<sub>2</sub>O.


==Procedure==
==Procedure==


# Extract DNA bearing piece of the gel using a razor blade.
# 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.
# Weight the gel piece in mg. This number is defined as V.
# Add 3V μL Gel Solubilization Buffer. Incubate 55 °C 10 min or until gel is completely dissolved.
# Add 3V gel solubilization buffer. Incubate 55 °C 10 min.
# After gel is completely dissolved, incubate 55 °C additional 5 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.
# Incubate RT 2 min.
# Add 1V isopropynol. Vortex and spin.
# Add 1V μL isopropynol. Mix and spin.
# Apply to PureLink gel extraction column. Spin in tabletop centrifuge max speed 1 min. Discard flowthrough.
# 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 500 μL wash buffer. Spin max speed 1 min. Discard flowthrough.
# Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough.
# Spin max speed 2 min to dry.
# To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube.
# Apply 100 μL sterile ddH<sub>2</sub>O to column membrane. Take care to not touch membrane with pipet tip.
# 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.
# Incubate RT 10 min.
# To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube.
# Spin max speed 2 min to elute.
# Quantify DNA sample by [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore].
# 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.
# Label side of tube with the following:
#* [DNA] in nM
#* "gel purified"
#* date
# Store at -20 °C.


==Notes==
==Notes==
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==References==
==References==
'''Relevant papers, books, and websites'''
[http://tools.invitrogen.com/content/sfs/manuals/purelink_quick_gel_extraction_kit_man.pdf Invitrogen Purelink Gel Extraction Kit manual].
<!-- If this protocol has papers or books associated with it, list those references here.-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
#[https://www.lifetechnologies.com/us/en/home.html Invitrogen Life Technologies]


==Contact==
==Contact==
Line 48: Line 46:


or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
==Digital Signature==
*'''SC 17:57, 25 July 2012 (EDT)''':


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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

  1. 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.
  2. Add 3V μL Gel Solubilization Buffer. Incubate 55 °C 10 min or until gel is completely dissolved.
  3. After gel is completely dissolved, incubate 55 °C additional 5 min.
  4. Incubate RT 2 min.
  5. Add 1V μL isopropynol. Mix and spin.
  6. 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.)
  7. Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough.
  8. To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube.
  9. Apply 50-100 μL elution buffer or sterile ddH2O to column membrane. Avoid touching membrane with tip. Incubate RT 10 min.
  10. To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube.
  11. Quantify DNA sample by quantifluore.
  12. Label side of tube with the following:
    • [DNA] in nM
    • "gel purified"
    • date
  13. Store at -20 °C.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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):
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