Protocols/Create
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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.