3 replies to this topic

[thekid]

Hello,

        I am trying to standardize a taqman assay using a probe of 24mer with Fam as reporter and Tamra as the quencher, the probe is from SIGMA and is at stock concentration of 100UM made with TE buffer.  The Calculated Tm of the probe is 65.3C and those of the primers are 63C. Am using the ABI Universal Taqman reagent with UNG for the assay. The conditions are 50C for 2 mins, 95C for 10mins and 35 cycles of 95C for 15sec and 60C for 1min.

I use diluted plasmids from copy number starting from 10 power 7 to 10 power 2 in a ABI 7500 machine. I have previously checked all the diluted plasmids and primers with SYBR green and they have given me r2 of 0.999 and efficiency of 98.9%. But when I use the same set of primers and diluted plasmids for the Taqman assay it does not work. There is no amplification see at all. When I run the products on a gel I see clear bands without any primer dimer or non specific bands.

I am using :

8]Taqman MasterMix 2x         12.5ul

    primer (20pmoles/ul)           1.0ul
    primer  (20pmoles/ul)          1.0ul
                        probe (20uM)                       0.2ul
    RNase-free H2O                  8.3 ul
                                                                    23.0ul
                       Plasmid                                 2.0ul
I have played with increasing and decreasing the probe concentration, anneling temperature went down to as low as 50C and as high as 62C with 2 degree increments every tube had good bands as observed on a gel but no amplification was seen on the plot.

Is it a problem with the probe?



thekid

[Anexum]

From what Ive seen of taqman. (presuming the same number of cycles between your gel and PCR) it could be that the 24mer is a bit too long for use as a probe. Ive been told that 20mer probes are bordering on too long. Its sadly one of the problems with Taqman.

SYBR green in my experience is only a little less accurate than Taqman. And if you can show specificity by PCR gel (and maybe northern blot if you wanted to be anal about it), its generally well accepted.

[thekid]

I agree that the limitations are 20mer's but I have worked with probes that are upto 25mers. What is more worse in the current situation is that I have reduced the anneal all the way to 50C and still there is no detection in the amplification plot. I noticed that in the multicomponent plot that the Tamra has a higher signal followed by Rox and Fam this I would assume is because the Probe has not bound to the plasmid and hence Fam signal is still being absorbed by Tamra and hence more of Tamra signal than Fam.

But I have already lowered the anneal to as low as 50C.

thekid

[Trof]

Few notes from various design tips
- usually they recomend Tm of the probe to be 5-10 deg higher than primers (mosty 10) - so hardly it can be as short as 20 bp without MGB or other modifiers
- no G at 5' end (basically less G the better)
- shorter than 30 bp
TAMRA is now a depricated quencher, but with singleplex FAM OK I guess


Nowadays companies usually design Taqman assays for free if you order oligos from them. I think it's better than try to design it on your own. Probes are expensive.