Hi Thyana,

I would be cautious with homebrew qPCR mixes, especially Sybr based. Sybr is not stable under all conditions, so you need to optimize the mix in this way. In addition you need to use the right concentrations, as Sybr Green is impairing polymerase activity in certain concentrations.

In the end I would not recommend to go this way as it might work nicely in one batch of mix but might not work as good in the next lot. In addition these 10.000x Sybr stocks are in most cases intended for stainign of Agarose gels so I am not sure if they are compatible with PCR mixes. Here I would rather consider to use a Sybr Green mastemrix and use the efforts for setup and analysis of experiments

Sven

Dear Thyana, be careful with homemade mix like @Sven said. But if you want to do it anyway ) I can provide You a good solution.

For 1 reaction (25ul)

1. Water up to 25 ul

2. Mg (50 mM stok) 1 ul, if 25 mM - 2 ul

3. dNTPs (10 mM mix) 0.25 ul

4. Forward primer 10-12 pmol (per reaction) or 500 nM (per liter, concentration).

5. Reverse primer - the same.

6. 10x PCR buffer (usually privided with Taq polymerase) 2.5 ul

7. 1x Sybr in DMSO - 1 ul. (waste residual mix of Sybr after each run, do not use it again)

8. Taq polymerase (5U/ul) 0.25 ul

9. DNA - 50 ng/reaction (for example)

it is a basic conditions. Yes, usually Sybr needs more Mg, but try provided protocol as a start protocol.

Next, prepare opt1 protocol with Mg 2-fold increasing and opt2 protocol with 2-fold promer decreasing. It is basic steps to optimize your reaction.

Please do it in temperature gradient (annealing/elongation step) at 56, 58, 60 , 62, 64 oC and run agarose gel after even You do not receive any curve.

Good luck.

I always use 100 pmol/ul stok of primers.

If you have 10000x Sybr, you can dilute it to 100x and than in 10 fold to receive 10x. After that you can save ) 100x at -20 and use some times.

Hi. This is the PCR buffer IV that I like to use in e.g. Rotorgene:

Final concentrations: 750 mM Tris buffer (pH 8,8 at room temp), 200 mM Ammonium sulphate, 0,1% Tween 20. Make 10x aliquots for the freezer and pr ml of 10x add 1 ul stock of Syto 9 (Thermo Fichser scientific ). Note This dye is much less inhibitory than Sybr Green, not expensive pr sample, and even makes better melt curves than EvaGreen. Perhaps of interest: Chapter High-Resolution Melt Analysis