I would like to amplify the cDNA of my protein of interest from the Baculovirus suspension. Actually, I would like to be sure that a specific mutation is well present.
Any recommendations?
First, you cannot amplify cDNA from bacaulovirus you need to do it from the host cell (Sf9). You can check for your mutation by PCR directly from your bacaulovirus solution. I don’t see why not. Colony PCR works well on intact bacteria and several species of yeast.
A quick search guides me it is a dsDNA virus? if your DNA is a part of baculoviral DNA then just isolate the particles from the suspension by (ultra) centrifugation (if they make cell free particles) and isolated DNA from them.
We tried to do a simple PCR using several volumes of baculo suspension. It didn't work. Moreover, we got an amplification in a PCR control using the purified corresponding vector. Do you think we have to extract the viral DNA prior to do the PCR? Or heat the PCR mix a bit longer before starting the cycles ?
Hi all,
Finally, we succeeded to amplify a band a the correct size after purifying the DNA from infected cell with a simple kit to do plasmid prep! Thank you for all your suggestions. Best. N
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