I am amplifying a gene from pET11a vector. Is the temperature for denaturation (95 C for chromosomal DNA generally) different for amplifying the gene from plasmid DNA?
You will have to use same temperature because plasmid also have same type of hydrogen bonding between nitrogenous basis as present in chromosomal DNA
Dear Suraj
Denaturation temperature for all DNA is same (95), it easy to amplify a gene from vector, you can dilute vector upto 1:100 or 1:1000, it depends on conc of plasmid. If you take high conc of plasmid as template you will also see some plasmid on gel with PCR product.
Suraj, there is no difference in the denaturation temperature used for plasmid (93-98 deg C depending on your polymerase). There is usually a shorter time used for the initial denaturation step of your PCR for melting plasmid DNA though, since it is much smaller than chromosomal DNA (30 sec to 1 min instead of 2-3 min).
There are no difference that I've noted, using 95 C for both cDNA and plasmid vectors work fine for me.
I agree that the denaturation temperature for all DNA is set at 95 .In the lab as a matter of course we perform PCR , RIBOTYPING and REALTIME PCR on clinical samples and this is the standardised temperature that is used .
Hope this helps.
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