Protocols/Create: Difference between revisions

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#Check your work by clicking the http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
#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.
#Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.
==Overview==
Replace this sentence with a brief description of the protocol and its goal.
==Materials==
For a 50 μL PCR reaction:
* 35 μL H<sub>2</sub>O
* 5 μL 10X PCR buffer
* 5 μL 2mM dNTPs (each)
* 1.5 μL 50mM MgCl<sub>2</sub>
* 1 μL 50μM sense primer
* 1 μL 50μM antisense primer
* 1 μL 5nM DNA template
* 0.5 μL TAQ DNA polyermerase
==Procedure==
# In a PCR tube, mix the components on ice in the order they are listed above.
# Perform thermocycling program
## 95 °C 5 min
## 95 °C 30 s
## T<sub>H</sub> 30 s
## 72 °C 1 min for each 1 kb PCR product
## Repeat steps 2-4 a total of 12-36 times (24 is standard).
## 72 °C 5 min
## 12 °C hold
==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]]
-->

Revision as of 11:18, 12 July 2012

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.

  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 http://openwetware.org/images/2/28/ShowPreviewButton.jpg button.
  6. Save the changes by clicking http://openwetware.org/images/7/71/SavePageButton.jpg ... and you're off.


Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL TAQ DNA polyermerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program
    1. 95 °C 5 min
    2. 95 °C 30 s
    3. TH 30 s
    4. 72 °C 1 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min
    7. 12 °C hold

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.


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