Protocols/Create: Difference between revisions

Revision as of 11:33, 12 July 2012

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
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.

This get you the DNA

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

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.

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

@@ Line 15: / Line 15: @@
 ==Procedure==
-# In a PCR tube, mix the components on ice in the order they are listed above.
+Collect a turbid sample of 10ml overnight growth
-# Perform thermocycling program
+# Centrifuge universal bottle for 10min 2600rpm
-## 95 °C 5 min
+# Discard supernatant
-## 95 °C 30 s
+# Resuspend cell pellets with 250μL of P1 Buffer by pipetting
-## T<sub>H</sub> 30 s
+# Transfer to sterile labelled eppendorf tube
-## 72 °C 1 min for each 1 kb PCR product
+# Add 250μL of P2 Lysing Buffer
-## Repeat steps 2-4 a total of 12-36 times (24 is standard).
+# Wait for 3-5 minutes (e.g. 3.5min)
-## 72 °C 5 min
+# Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
-## 12 °C hold
+# 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==