| | Re: Real Time PCR product size
This is all dependent on what type of analysis you intend to do, to tell you the truth.
A. Are you looking to do simple absence or presence ? IF so Sybr is just fine and you can simply shotgun it with a touchdown PCR if you don't know your annealing temps... just make sure you take your BP amplicon and divide this by 25 to reach your annealing hold time.
B. Are you doing relative Quantification? This is a loaded question because there are two types of RQ. The first type as listed above RELIES on perfect efficiency which means that with large amplicons like this, you better be 1. God's gift to QPCR, 2. come with natural ability that makes Tiger Woods cry, 3. Normal like the rest of folk, Better use the other relative quant and not this method. If you are #3, then I would suggest running a housekeeping gene standard curve and a target gene standard curve, which will then be your normalizing factor for all Cp values after this point. But enough of that!
3. Genotyping ; melting curves, melting curves, melting curves. This is relatively easy as you can simply add either a Tm melt or an HRM analysis at the end of your sybr green run. This is also another option for presence or absence.
4. Basic Quantitation : You can do Sybr Green but you can also run the probes method which I personally do, but you would need some extra $$ initially. Either way, just be sure to run a Standard curve - you could run a 10x starting from 100ng, 10ng, etc. This will also establish your efficiency and linearity. Be sure your professor/boss isn't watching at this point because this also shows just how wonderful or stupendously bad your pipetting technique is.