syber green with real time PCR - (Jul/31/2006 )
does anyone have a protocol for usig syber green instead of taq man probes in real time PCR ????
I do it that way
a specific question will be easier to answer, though...my protocol is optimized for my system
have you read ABI's User Bulletin #2? there are many suggestions
im using ( or well be using to be specific ) a real tome PCR stratagene MX 3000 to detect
carriers of duchenne muscle dystrophy but all the protocols i found so far depend on taqman
probe ( expensive ) thats why im asking about cyber green
i didnt read what u asked me about ,,,, what is it?
go to this website
type "user bulletin #2" into the search box. download the pdf (it's free) and read it. there's tons of great stuff in there if you're just getting started. it's a little hard to read, but it gets better. also, if you call (or email) Stratagene's tech support and ask them, they will send a rep to your area, sometimes even to your lab, who will sit down and explain the whole thing. or, they can set you up with an online tutorial, or just answer your questions via phone/email
OH, hey, just so we're all on the same page...if you already know this, I'm sorry...but the major difference, is that you have to optimize your own primers with SYBR green. you also must do melting curve analysis with your runs
thanks a million
i didnt know that i have to optimize my primers with cyber greeen
u see im a beginner in the field of molecular genetics .... do u know any sites that deal with he basics of moleculargnetics ??/
I didn't bookmark it at the time, of course...but I wanted a refresher a few years ago and I went to google and typed in 'genetics tutorial'
obviously I got pages of garbage...but with a little sifting, I came across notes posted for an upper level undergraduate course on genetics. they were very good and included pictures too. I would recommend for you to do something similar, just to be up on the theory
if you want actual application notes (like, how to make DNA, how to run a protein gel, etc) start with Sambrook's 3-volume Molecular Cloning. It's a little old, but is admittedly the bible even today.
lula, what is your background and education? and, is someone training you or do you have to figure this out yourself?
no ...im trainning myself based on some basics i catched from a collegue ,,,,i have a master in genetics ,,,but the molecular lab work is somewhat a new teritory for us so we are trying to develope our lab as much as we can
u know we didnt even do a southern blot yet
and sambrook book that u consider as a very important book ,,,is not available at alll to me ...i was hoping that i could find an online virsion of it
forgot to tell u
we did RT-PCR and PCR RFLP ,,,nested PCR ,,,multiplex PCR ...but thats about it
oh, if you've done RT-PCR you're almost there and you know most of what you need to know to make real-time work. and if you have a master's in genetics you're ahead of the game. I wasn't sure where to start, that's why I asked...hope you weren't offended?
I use SYBR, and basically when you start out it's a bit more intensive. you must very carefully design your primers (but you should have the skills you need from RT-PCR). again, ABI's website is an excellent tool; you can find much of what you want at www.ambion.com, and also I've had some good luck with BioRad's website - even if you don't use their products, they have some good technical literature.
once you have designed your primers, you have to basically titrate them simultaneously with template to determine which concentration of each will give you the best efficiency. then, you're ready to roll, the hard part's done and you can begin going through samples like hotcakes. you can find the details you need to set this up and do the calculations in User Bulletin #2, or at least get an idea of the general flow of the setup experiments.
and, there are always many of us here who can help out if you get stuck