Extraction of adenovirus DNA from cell culture for PCR - protocol (Jan/31/2005 )
Does anyone have the protocol of PCR for cell culture (adevovirus) ? Please help me. Thanks.
"the extraction of adenovirus DNA from cell culture for PCR"
Our lab's viral DNA (Herpesvirus) has us take several flasks of infected cells. Take S/N and spin down cell debris (1000 rpm ~5min) then take S/N (don't get any cell pellet) Spin at 12000 rpm for 2 hours. You get a little pellet (At least with herpes we do).Resuspend in a bit of buffer. Treat with SDS and Proteinase K. After that it is just a phenol chloroform extraction with ethanol precipitation. We do a lot of PCR from viral DNA and this method works for us. I don't have the protocol with me right now so this is kinda vague, but I hope that helps ya !
I had a question regarding the confirmation of Ad5 constructs.
I use Ad5 to amplify constructs, and I usually get a yield of 1-6 x 10E12 vp/ml. I start off using 1 T75 flask, and go through 2, 6, 16 T-150 plates, and finally 2 Nunc cell factories. I use the CsCl virus recovery system, and its worked fine for me.
I'd like to know, if there is any way for me to confirm that I have the right adenovirus construct during the course of amplification. i.e , I get to sequence the maxi prep DNA (before Pac I digest), and use that to carry on the process. The confirmation happens when I extract Ad- DNA using Pro K and Phenol-choloroform extraction, and then sequencing the DNA to check if I have the right Adenovirus construct.
Is there any way that I can confirm the sequence of the Ad5 at some stage in between, so that I'm 100% sure I am proceeding with the right stuff?