My only concern is that when I pick a primer pair from RTPrimerDB (with a Ta of 60°C) and put it into Primer-Blast without filling those boxes, the Tm fall between 50 and 55°C. If I use 2.5mM as "Concentration of divalent cations" and 0.2mM as "Concentrations of dNTPs" (as far as I know, these could be realistic values in the PCR-tube), Tm rises of about 7°C. Likewise, If I design my primer pair using a Tm of 60°C, the actual Tm will be 67°C after filling those boxes.
Thus, I wonder whether the rise in the Tm could affect my qPCR and lead to unspecific amplification or it is of little importance and even a slighlty higher Tm (67-68°C) works well with a Ta of 60°C.
I'm experiencing some problems using Primer-Blast to design prmer pairs. In particular, I don't know how to fill the boxes "Concentration of divalent cations" and "Concentrations of dNTPs", because I haven't found these values in datasheets of many SYBR Green PCR master mixes. I wonder whether there are commonly used fixed values for these parameters. As far as I understand, these values are important to calculate primer Tm, right?
Yet another question. Should primer Tm be slighlty higher than annealing temperature (for example, 5°C higher), or should it be equal to annealing temperature?
my boss told me to start T cell cloning experiments but I don't manage to obtain enough feeder cells.
Because we don't have an irradiator, I treat 10^7 PBMcs with 500ul RPMI+mitomycin C (final concentration 25ug/ml).
After incubating them at 37°C 5%CO2 for 30', I wash at least 5 times with RPMI.
Finally, I count the cells and I find no more than 10^6 PBMCs!!
Could anyone give me some advice to improve the yield and tell me what's wrong with this protocol?
It would be wonderful to learn from your experience!!
Thanks in advance,