Protocols/Template: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
mNo edit summary
 
(73 intermediate revisions by 8 users not shown)
Line 1: Line 1:
<center>
{|style="width: 48em; background: #FF6677;"
| align="center"|'''This page is a template and should not be edited.'''<br><span style="font-size:90%">Click [http://openwetware.org/index.php?title={{PAGENAME}}&action=edit here], copy the source, and paste it into your page.</span>
|}
</center>
'''Interested in posting a protocol on OpenWetWare?  Here is a template to help you do so.''' 
'''Click the view source tab and copy everything below this line.  Paste it into your new protocol page.  Then replace the text in this page with your own protocol.  Feel free to add or delete sections as appropriate.'''
[[Category:Template]]
<!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->
==Overview==
==Overview==
This protocol describes the procedure for streaking bacteria on an Agar plate.
 
Replace this sentence with a brief description of the protocol and its goal.


==Materials==
==Materials==
*Agar plate(s)
 
*Bacteria sample from old plate
For a 50 μL PCR reaction:
*Inoculation loop
 
*Bunsen Burner
* 35 μL H<sub>2</sub>O
*70% Ethanol
* 5 μL 10X PCR buffer
*Parafilm
* 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==
==Procedure==
#Wipe down the bench with 70% EtOH and start a Bunsen burner flame.
# In a PCR tube, mix the components on ice in the order they are listed above.
#Label new agar plate(s), on the bottom, with your initials, the date and strain of bacteria, and prepare plastic beaker for containing the disposable at the side#Step 3
# Perform thermocycling program
#Flame until red to disinfect the inoculation loop, from top toward the loop, and allow it to cool down by inserting to loop into the agar at a corner of the new agar plate.
## 95 °C 5 min
#(work by the flame) Open the old streaking plate, wipe down water vapor on the cover with Kimwipes, and take one (1) pure culture with the loop.
## 95 °C 30 s
#Slide the loop over the new agar plate. First start circulating ~10 times at a corner of the streaking area, followed by moving the loop in zigzag patterns to spread the colony on all 4 quadrants over the plate.  Between each spread, flame the loop to disinfect and allow it to cool down. Cross over the previous quadrant 2~3 times and avoid cross over the previous quadrant more than once or into any other quadrants for the last streak.
## T<sub>H</sub> 30 s
#Cover the new dish and seal the edge of the old plate with parafilm.
## 72 °C 1 min for each 1 kb PCR product
#Incubate the new plate @37oC in a upside-down position (to allow water to drip onto the cover) overnight (~24hrs)
## Repeat steps 2-4 a total of 12-36 times (24 is standard).
#Bleach and wipe down the bench with 70% EtOH again at the end.
## 72 °C 5 min
#Take the plate(s) out from the incubator on the next day and store it inside the fridge with parafilm sealing the edge. (good for a month)
## 12 °C hold
#Bleach the old dish (entirely cover the plate) and seal with parafilm. Dispose it in the biohazard trash.


==Notes==
==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==
==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]].


==Contact==
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
Sylvanus.


or instead, [[Talk:{{Sylvanus_Y.K._Lee}}|discuss this protocol]].
[[Category:Escherichia coli]]
<!-- Move the relevant categories above this line to tag your protocol with the label
<!-- 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 14:27, 18 June 2012

This page is a template and should not be edited.
Click here, copy the source, and paste it into your page.



Interested in posting a protocol on OpenWetWare? Here is a template to help you do so.

Click the view source tab and copy everything below this line. Paste it into your new protocol page. Then replace the text in this page with your own protocol. Feel free to add or delete sections as appropriate.

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.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Protocols/Template&oldid=608315"