tag:blogger.com,1999:blog-5522710872110316695.post2990896374955181055..comments2007-09-28T12:41:20.958+02:00Comments on Anton Dohrn Café: On real time PCR, cDNA kits etc.Anton Dohrn caféhttp://www.blogger.com/profile/10363582350563112650noreply@blogger.comBlogger2125tag:blogger.com,1999:blog-5522710872110316695.post-24959191857499901072007-07-20T09:39:00.000+02:002007-07-20T09:39:00.000+02:00Well as I already said, 8ul is a small volume so i...Well as I already said, 8ul is a small volume so it makes your experiments to be more error prone (hence the need for replicates). to stay on the safe side I do 10ul rxs and it's been working very well.<BR/><BR/>As for the Genomic DNA wipeout, I still use it, just beacuse it's in teh kit and it takes 2 min to do it. However when prepping DNA with quiagen columns (this is how I do it nowadays), I do have to do a DNAse step during the procedure (even if it is considered optional with the all prep DNA/RNA kit). So the DNA wipeout reagent alone is not enough to emove all genomic DNA from my samples. So in my experience it is "insufficient" more than "useless".<BR/><BR/>Gabriele AmoreAnton Dohrn caféhttps://www.blogger.com/profile/10363582350563112650noreply@blogger.comtag:blogger.com,1999:blog-5522710872110316695.post-42170368944174848482007-07-18T10:43:00.000+02:002007-07-18T10:43:00.000+02:00hey !A comment on the last post. Thanks !Enrique.-...hey !<BR/>A comment on the last post. Thanks !<BR/><BR/>Enrique.-<BR/><BR/>---------------------------------------------------------------------<BR/><BR/>Comment on “On real time PCR, cDNA kits etc.”<BR/><BR/>Really good to know! BUT I’ll stress the point<BR/>of pipeting small volumes and the big potential<BR/>variability that comes with it. Be careful and include two (three?) replicates.<BR/>The only point I don’t agree with is on how<BR/>useless it can be to use the ‘genomic DNA wipeout<BR/>reagent’. Are you saying that you don’t use it<BR/>because you have checked that there’s no DNA<BR/>contamination after RNA extraction?<BR/>I’ll say that it depends on how I do the<BR/>extraction (i.e. Kit, eurozol, etc..). But it<BR/>applies even if I trust the 2 step extraction kit<BR/>(DNA and RNA) of the QuantiTect cDNA kit from<BR/>Quiagen. Unless I want to run always a control on<BR/>my qPCR plate with RNA in stead of cDNA, the<BR/>DNAse step should be done. Not to include that<BR/>control can be quite problematic in order to be confident on positive results.<BR/>An then, what happens if I actually find a<BR/>positive result on my RNA control? Re-do the<BR/>whole cDNA? In my case I usually have a low yield<BR/>of RNA, so I can’t manage to use it directly as a control material.<BR/>My two cents on the subject. At the end all<BR/>cookers have their special recipe…right?<BR/><BR/>Cheers !<BR/><BR/>Enrique Arboleda<BR/>______________________________<BR/><BR/>Enrique Arboleda<BR/> Biochemistry and Molecular Biology Laboratory<BR/> Stazione Zoologica "Anton Dohrn"<BR/> Villa Comunale 1 (80121)<BR/> Napoli - ITALY<BR/> e-mail: enrique.arboleda@szn.it<BR/> Tel. (+39) 081 583-3255<BR/> Fax (+39) 081 764-1355Anton Dohrn caféhttps://www.blogger.com/profile/10363582350563112650noreply@blogger.com