Protocols/Template: Difference between revisions
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'''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.''' | '''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! --> | <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> | ||
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==Materials== | ==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== | ==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== | ==Notes== | ||
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==References== | ==References== | ||
'''Relevant papers and books''' | '''Relevant papers and books''' | ||
<!-- If this protocol has papers or books associated with it, list those references here. | <!-- 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> | <biblio> | ||
#Goldbeter-PNAS-1981 pmid=6947258 | #Goldbeter-PNAS-1981 pmid=6947258 | ||
#Jacob-JMB-1961 pmid=13718526 | #Jacob-JMB-1961 pmid=13718526 | ||
#Ptashne-Genetic-Switch isbn=0879697164 | #Ptashne-Genetic-Switch isbn=0879697164 | ||
</biblio> | </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== | ==Contact== | ||
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
- 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
- TH 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 '''*~~~~''': 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.
