Plasmid PCR

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chinna
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Plasmid PCR

Post by chinna » Wed Aug 03, 2005 5:37 pm

If i am having gene of interest present on Plasmid. And i want to do PCR of that particular gene. How shud i proceed for PCR??? I mean do we hav to get Plasmid linearized prior to PCR or it can work with normal cycles of PCR having profile of Denaturation,annealing and extension.

cauguama
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Post by cauguama » Wed Aug 03, 2005 6:56 pm

When you run the PCR reaction, your plasmid DNA will denature to ssDNA and be available for binding to your primers. So turning it into linear DNA is not required. When figuring your temperatures for the reactions, it helps to take into account the sequence of your primers so as to know the correct melting/annealing/extension temperatures. The most common ones are (give or take a degree or two) 90C-2min, 90C-15sec, 56C-15sec, 72C-30sec to 2min, repeat 20-30 times (excepting the first 90C-2min, then finally a 72C extension for 5-10min; then you're done!

Daniel Tillett
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Post by Daniel Tillett » Wed Aug 03, 2005 11:40 pm

It can help if you denature the plasmid before doing the PCR. If you do manual hot starts then denature the plasmid by heating the reaction to 98C for 3 min before adding the Taq enzyme.

Daniel

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