In qPCR, I am using sometime 6 microL Syber with 1 miro cDNA, 2 micro F/R primers and 1 micro water in a 10 microL reaction mix together. I also use 12 micro Syber miro cDNA, 2 micro F/R primers and 5 micro water in a 20 microL reaction mix together. I am not sure which conbination will give the expected results.
Hi,
which is the suppliers recommendation? In most protocols you will find a guidance for different reaction volumes. Just keep in mind, that the volume need to fit with your instrument e.g. you cannot do a 5µl reaction in a 96 well plate.
In general it would be better to get concentration for the used reagents as saying 2µl does not give any info without concentration
Sven
Sven Reister is correct that you should make sure the final concentration (not volumes used) needs to be identical for your different reactions. If the SYBR is the same concentration, then adding 1/2 of the reaction volume of SYBR to each reaction gives the final concentration.
As a general rule, larger reaction volumes are better than smaller (less variation due to evaporation, poor pipetting skills, etc.).
Presumably when you say "SYBR" you actually mean "PCR master mix with SYBR green included", because you haven't listed any reaction buffer components anywhere.
If this is the case, then your master mix should be supplied as a concentrated stock with a very clearly listed dilution factor: 2x stock is typical for qPCR buffer in my experience, in which case you should always dilute it 2-fold in your final volume (so 5ul master mix in 10ul PCR, 10ul master mix in a 20ul PCR, etc).
This dilution is the optimum, and indeed really the ONLY dilution factor you should use: there is no justifiable reason to use a prepared pre-mix at higher or lower dilution than specified by that pre-mix.
I find it unlikely that your master mix is a 1.66x stock (implied by use of 6ul in 10ul PCR), so I would humbly suggest that both of your suggested conditions are probably wrong.
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