Protocols/Create: Difference between revisions
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==Creating a New Protocol== | ==Creating a New Protocol== | ||
#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]] | |||
#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" /> | |||
#Click on the create this page link. | |||
#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.''' | |||
#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button. | |||
#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off. | |||
==Miniprep== | |||
This gets you the DNA | |||
==Procedure== | |||
Collect a turbid sample of 10ml overnight growth | |||
# 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 | |||
# Wait for 1 minute | |||
# Centrifuge for 1 minute (13K rpm) to elute | |||
# 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! | |||
#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. | |||
==References== | |||
'''Relevant papers and books''' | |||
<!-- If this protocol has papers or books associated with it, list those references here.--> | |||
<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently. | |||
<biblio> | |||
#Goldbeter-PNAS-1981 pmid=6947258 | |||
#Jacob-JMB-1961 pmid=13718526 | |||
#Ptashne-Genetic-Switch isbn=0879697164 | |||
</biblio>--> | |||
<!-- Try the [[Template:FormatRef|FormatRef template]]--> | |||
#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258 | |||
#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526 | |||
#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164 | |||
==Contact== | |||
*Who has experience with this protocol? | |||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | |||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | |||
<!-- Move the relevant categories above this line to tag your protocol with the label | |||
[[Category:Protocol]] | |||
[[Category:Needs attention]] | |||
[[Category:In vitro]] | |||
[[Category:In vivo]] | |||
[[Category:In silico]] | |||
[[Category:DNA]] | |||
[[Category:RNA]] | |||
[[Category:Protein]] | |||
[[Category:Chemical]] | |||
[[Category:Escherichia coli]] | |||
[[Category:Yeast]] | |||
--> |
Latest revision as of 18:50, 18 March 2013
Creating a New Protocol
- In the search box on the lefthand side navigation bar, type
Protocol name
. If it is a protocol specific to your lab, typeLabName:Protocol name
(be sure and replaceLabName
with your PI's last name likeKnight
orSmolke
- 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.
- Click on the create this page link.
- View and copy the source from the protocols template into your new page and begin editing. Make sure you are not editing this page.
- Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
- Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.
Miniprep
This gets you the DNA
Procedure
Collect a turbid sample of 10ml overnight growth
- 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
- Wait for 1 minute
- Centrifuge for 1 minute (13K rpm) to elute
- 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!
- 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 and books
- Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
- Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
- 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.