SYBR green PCR - real time (Oct/30/2007 )
I'm trying to do some real time PCR but we dont want to spend loads of money in any specific real time SYBR green kit. We are just trying some machines at the moment to decide whether we buy one or not, we dont have much money and it will take me forever to convince my managers that we need the reagents (longer than we have the demo units for sure).
anyway, after all this moaning, i've been trying standard PCR reagents (either invitrogen Taq, or ABI AmpliTaq Gold) with SYBRgreen 1x in the reaction mix. I see high fluorescence from the very first cycle and this does not increase with cycle no. but decreases (quenching?) As I see no fluorescence at all in my no template control, i have the feeling my template (genomic DNA) is binding all the SYBR green.
Also, cant make much sense from the melting curves, but that might just be that i dont know the software.
any help, suggestions, ideas would be more than welcome.
I think when u add sybergreen externally u need to add more Magnesium.
U can standardize with different Magnesium concentrations.