DeRosa:Protocols/Rolling circle amplification
< DeRosa:Protocols(Difference between revisions)
Maureen McKeague (Talk | contribs)
(New page: ==Overview== Replace this sentence with a brief description of the protocol and its goal. ==Materials== * Use Templiphi Kit ==Procedure== #Transfer 5 μl aliquots of sample buffer to a...)
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Replace this sentence with a brief description of the protocol and its goal.
- Use Templiphi Kit
- Transfer 5 μl aliquots of sample buffer to appropriate PCR tubes.
- Transfer samples to the dispensed sample buffer. For bacterial colonies, transfer a small portion of a colony directly to the dispensed sample buffer. A gentle touch of a gel pipette tip on the colony surface provides sufficient starting material. Avoid transferring agar from the plate or excess cell material into the TempliPhi reaction. Transfer only a small portion of a colony (by stirring the tip with the some of the colony on it around in the tube). Use a DIFFERENT tip for each colony.
- Denature the sample. After the sample is added to sample buffer, seal the reaction tubes with an appropriate lid. Heat at 95°C for 3 minutes, and then cool to room temperature or 4°C. The rate of cooling is not critical.
- Prepare TempliPhi premix. In a separate tube, combine 5 μl of reaction buffer and 0.2 μl enzyme mix for each TempliPhi reaction. It is convenient to make a master mix sufficient for the required number of TempliPhi reactions just prior to use. Once made, the master mix must be used the same day and not stored for future use.
- Transfer 5 μl of the TempliPhi premix to the cooled, denatured sample prepared in step 3.
- Incubate at 30°C for 18 hours.
- Heat-inactivate the enzyme by incubating at 65°C for 10 minutes.
Cool to 4°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.