Corum:Gel Purification: Difference between revisions

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==Overview==
==Overview==


Vector construction by ligation of dsDNA linker into a dsDNA vector backbone with compatible restriction sites.
Gel extraction with [http://products.invitrogen.com/ivgn/product/K210012 Invitrogen Purelink Gel Extraction Kit].


==Materials==
==Materials==


* ssDNA linker oligonucleotides OR ssDNA primer oligonucleotides and linker DNA template
* DNA embedded in gel.
* PCR reaction components
* Gel electrophoresis components
* Gel extraction kit
* PCR purification kit
* Ligation reaction components
* Transformation components
* Miniprep kit


==Procedure==
==Procedure==


# Design linker and vector backbone with compatible overhanging restriction sites.
# 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.
# Construct linker by
# Add 3V μL Gel Solubilization Buffer. Incubate 55 °C 10 min or until gel is completely dissolved.
#* Hybridization of ssDNA oligonucleotides (<100bp)
# After gel is completely dissolved, incubate 55 °C additional 5 min.
#* OR PCR amplification of linker DNA template with ssDNA oligonucleotide primers (>100bp) followed directly by double digestion with restriction enzymes (heat inactivate 80 °C 20 min) and PCR purification of the digestion product. Elute in 100 μL sterile H<sub>2</sub>O.
# Incubate RT 2 min.
# In parallel, construct vector backbone by double digestion with restriction enzymes (30 μL reaction volume), followed by gel extraction of the backbone, leaving the cut-out piece behind. Elute in 100 μL sterile H<sub>2</sub>O.
# Add 1V μL isopropynol. Mix and spin.
# Perform ligation on the linker and vector backbone.
# 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.)
# Transform 3 μL ligation product into competent cells.
# Apply 700 μL wash buffer to column. Centrifuge max speed 1 min. Discard flowthrough.
# Grow 4-6 parallel overnight minicultures from competent cells.
# To dry, centrifuge max speed 3 min. Place column in a new 1.5 mL tube.
# Perform miniprep plasmid isolation on the minicultures.
# Apply 50-100 μL elution buffer or H<sub>2</sub>O to column. Incubate RT 10 min.
# Sequence the miniprep products.
# To elute, centrifuge 1 min max speed. Discard column. DNA is now in bottom of 1.5 ML tube.
# Verify the constructs by comparing the expected linker sequence with the actual linker sequence. Retain good clones. Discard bad clones.
# Quantify DNA sample by [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore].
# 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].
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==Contact==
==Contact==
Line 47: Line 43:


==Digital Signature==
==Digital Signature==
*'''SC 18:00, 28 June 2012 (EDT)''':
*'''SC 17:57, 25 July 2012 (EDT)''':


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Revision as of 14:57, 25 July 2012

Overview

Gel extraction with Invitrogen Purelink Gel Extraction Kit.

Materials

  • DNA embedded in gel.

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 H2O to column. 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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