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Step 7: Rolling-Circle Amplification
Now that the plasmid is free of the cell, we need to amplify (make many, many copies of) just the fragment-containing pUC18 plasmid. This is achieved by a reaction called rolling-circle amplification, or RCA. If you are familiar with PCR (the polymerase chain reaction), then it may help you to know that RCA is very similar. The end result of RCA will be millions of snowflake-like structures, each containing several copies of the plasmid DNA.
New strands of DNA matching the DNA in the plasmid are formed in connected arcs as polymerase "rolls" around the circular plasmid (center ring).
To prepare the reaction, 10 µl of Amersham TempliPhi reagent is added to each of the 384 wells containing only 2 µl of the plasmid. The TemliPhi reagent consists of
- dNTPs (the four nucleotides, A, T, G, and C)
- phi29 polymerase (an enzyme that adds nucleotides to a strand of DNA)
- random primers (which give polymerase a place to start building)
The reaction is incubated for 18 hours at a constant 30°C.
An incubator is used for the RCA reaction.
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