Protocols/Create

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#REDIRECT [[Marek: Freeze-down/Thaw]]
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==Creating a New Protocol==
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#In the search box on the lefthand side navigation bar, type <code>Protocol name</code>.  If it is a protocol specific to your lab, type <code>LabName:Protocol name</code> (be sure and replace <code>LabName</code> with your PI's last name like <code>Knight</code> or <code>Smolke</code> [[Image:Searchbox.png|right]]
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#You should see a page come up with a message saying '''"There is no page titled "Protocol name".''' You can create this page." If you don't see this message then that means a page with that name already exists.  You'll need to choose a different protocol name.  [[Image:SearchforProtocol.tiff|450px|left]] <br style="clear:both" />
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#Click on the create this page link.
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#View and copy the source from the [[Protocols/Template|'''protocols template''']] into your new page and begin editing. '''Make sure you are not editing this page.'''
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#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
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#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.
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 +
 
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==Miniprep==
 +
 
 +
This get you the DNA
 +
 
 +
 
 +
 
 +
==Procedure==
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Collect a turbid sample of 10ml overnight growth
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# Centrifuge universal bottle for 10min 2600rpm
 +
# Discard supernatant
 +
# Resuspend cell pellets with 250μL of P1 Buffer by pipetting
 +
# Transfer to sterile labelled eppendorf tube
 +
# Add 250μL of P2 Lysing Buffer
 +
# Wait for 3-5 minutes (e.g. 3.5min)
 +
# Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
 +
# Centrifuge sample at 13K rpm for 10 minutes
 +
# Add supernatent to a nickle filter (ensure not solids)
 +
# Centrifuge for 1 minute (13K rpm) and discard flowthrough
 +
# Add 500μL PB to wash filter
 +
# Centrifuge for 1 minute (13K rpm) and discard flowthrough
 +
# Add 750μL PE (with Ethanol) for final wash
 +
# Centrifuge for 1 minute (13K rpm) and discard flowthrough
 +
# Centrifuge for 1 additional minute (13K rpm) and discard flowthrough
 +
# Transfer filter to sterile labelled eppendorf tube
 +
# Add 30μL Nuclease free water to centre of column
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# Wait for 1 minute
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# Centrifuge for 1 minute (13K rpm) to elute
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# Store sample on ice or in -20°C
 +
 
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==Notes==
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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 <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==References==
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'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here.-->
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<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>-->
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<!-- Try the [[Template:FormatRef|FormatRef template]]-->
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#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
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#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
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#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164
 +
 
 +
==Contact==
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*Who has experience with this protocol?
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
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<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
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 +
<!-- Move the relevant categories above this line to tag your protocol with the label
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[[Category:Protocol]]
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[[Category:Needs attention]]
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[[Category:In vitro]]
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[[Category:In vivo]]
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[[Category:In silico]]
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[[Category:DNA]]
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[[Category:RNA]]
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[[Category:Protein]]
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[[Category:Chemical]]
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[[Category:Escherichia coli]]
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[[Category:Yeast]]
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-->

Revision as of 14:33, 12 July 2012

Contents

Creating a New Protocol

  1. In the search box on the lefthand side navigation bar, type Protocol name. If it is a protocol specific to your lab, type LabName:Protocol name (be sure and replace LabName with your PI's last name like Knight or Smolke
  2. You should see a page come up with a message saying "There is no page titled "Protocol name". You can create this page." If you don't see this message then that means a page with that name already exists. You'll need to choose a different protocol name. Image:SearchforProtocol.tiff
  3. Click on the create this page link.
  4. View and copy the source from the protocols template into your new page and begin editing. Make sure you are not editing this page.
  5. Check your work by clicking the ShowPreviewButton.jpg button.
  6. Save the changes by clicking SavePageButton.jpg ... and you're off.


Miniprep

This get you the DNA


Procedure

Collect a turbid sample of 10ml overnight growth

  1. Centrifuge universal bottle for 10min 2600rpm
  2. Discard supernatant
  3. Resuspend cell pellets with 250μL of P1 Buffer by pipetting
  4. Transfer to sterile labelled eppendorf tube
  5. Add 250μL of P2 Lysing Buffer
  6. Wait for 3-5 minutes (e.g. 3.5min)
  7. Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
  8. Centrifuge sample at 13K rpm for 10 minutes
  9. Add supernatent to a nickle filter (ensure not solids)
  10. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  11. Add 500μL PB to wash filter
  12. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  13. Add 750μL PE (with Ethanol) for final wash
  14. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  15. Centrifuge for 1 additional minute (13K rpm) and discard flowthrough
  16. Transfer filter to sterile labelled eppendorf tube
  17. Add 30μL Nuclease free water to centre of column
  18. Wait for 1 minute
  19. Centrifuge for 1 minute (13K rpm) to elute
  20. Store sample on ice or in -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

Relevant papers and books

  1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


Retrieved from "http://www.openwetware.org/wiki/Protocols/Create"
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