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, type
LabName:Protocol name(be sure and replace
LabNamewith your PI's last name like
- 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. Image:SearchforProtocol.tiff
- 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 button.
- Save the changes by clicking ... and you're off.
This get you the DNA
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
- 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
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
- Who has experience with this protocol?
or instead, discuss this protocol.