What is the start and final concentration of you primers in master mix reaction? Also, in cycling stage what are the optimal temperature and the time for sybr green qPCR reaction?
In my experience with Roche's Lightcycler instrument I have always used the following mix and never had problems :
SYBR green master mix 10X (2uL) (Roche)
MgCl2+ (1.6uL) (Roche)
Each primer (forward and reverse) (2uL of 5uM stock solutions) ==> final concentration (0.5uM)
Graded water up to a final volume of 16uL (8.4uL)
Finally, I add 4uL of template to obtain a mix in a final volume of 20uL x capillary.
I have also tried the BioRad iTaq Fast SYBR green master mix and it works quite well with LightCycler. You only need to make some adjustments for the mix and don't need to add MgCl2+, since it is already present in the mix. I adapted the mix as follows:
iTaq SYBR green master mix (BioRad) (2X) (10uL)
Same as above for each primer set (2uL each)
Graded water up to 15uL (just 1uL)
Template in a final volume of 5uL.
PS. The concentration of the cDNA template has to be determined empirically, depending on the purity or other factors. Usually, a final concentration of 20nM/uL would be ok (final volume = 100nM in the latter example), but it also depends. Remember, both master mixes are hot start and require an initial step of 95°C for at least 1'30" to activate.
The annealing temperature you indicated is in the usual range. Optimally, higher temperatures are more stringent and would decrease the possibility of dimerization. Consider this when preparing your primers.
I hope this is helpful and...good luck with your experiments.
Alessandro Castorina, PhD
I routinely use 0.5 μl of each of the 10 μM forward and reverse gene-specific primers in a final volume of 10 μl. The PCR is carried out either in Roche's LightCycler or ABI7500. I aim to have an annealing temperature of 58-60°C and hold it at this temperature for 10 sec. I hope this helps.
In my experience with Roche's Lightcycler instrument I have always used the following mix and never had problems :
SYBR green master mix 10X (2uL) (Roche)
MgCl2+ (1.6uL) (Roche)
Each primer (forward and reverse) (2uL of 5uM stock solutions) ==> final concentration (0.5uM)
Graded water up to a final volume of 16uL (8.4uL)
Finally, I add 4uL of template to obtain a mix in a final volume of 20uL x capillary.
I have also tried the BioRad iTaq Fast SYBR green master mix and it works quite well with LightCycler. You only need to make some adjustments for the mix and don't need to add MgCl2+, since it is already present in the mix. I adapted the mix as follows:
iTaq SYBR green master mix (BioRad) (2X) (10uL)
Same as above for each primer set (2uL each)
Graded water up to 15uL (just 1uL)
Template in a final volume of 5uL.
PS. The concentration of the cDNA template has to be determined empirically, depending on the purity or other factors. Usually, a final concentration of 20nM/uL would be ok (final volume = 100nM in the latter example), but it also depends. Remember, both master mixes are hot start and require an initial step of 95°C for at least 1'30" to activate.
The annealing temperature you indicated is in the usual range. Optimally, higher temperatures are more stringent and would decrease the possibility of dimerization. Consider this when preparing your primers.
I hope this is helpful and...good luck with your experiments.
Alessandro Castorina, PhD
Primer concentrations for a qPCR using SYBR Green may vary a great deal. You may try to aim for a final concentration of 0.2 to 1.0 µM. Using a Roche SYBR Green kit in a LightCycler I get good results using a final concentration of 0.3 µM. In terms of times for each step, try 1 to 5 seconds for the denaturation step, the more GC-rich the template, the longer denaturation time is required. For the annealing step, aim for 5-20 seconds. Longer annealing times may allow for greater precision when it comes to target amplicon quantification. The extension time will vary according to the amplicon size. As qPCR product sizes are generally small, this will be quite short as well. Aim for about 1 second per 25 bp.
As far as temperatures are concerned, 95°C is usually used for the denaturation step. The temperature used during the annealing step depends on the primers used. A good place to start would be 5°C below the Tm of the primers. Lastly, most extensions are done at 72°C unless the manufacturer uses a different enzyme.
Hope this helps you and good luck with those amplifications.
Tnx all.
It was very helpful. I am using 7500 fast AB real time PCR. I am using the 50 nm primer concentrations and till now a got rather well results, But now I encounter a small problem. I came across a gene that obviously is low abundant in my tissue and I cant get the right dilution and right amplification
- Badges
- Science method