- August 3, 2005 at 5:37 pm #1588chinnaParticipant
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.
- August 3, 2005 at 6:56 pm #27997cauguamaParticipant
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!
- August 3, 2005 at 11:40 pm #28003
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