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borriello.87's Content

There have been 4 items by borriello.87 (Search limited from 18-April 20)


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#131693 Help with primer design

Posted by borriello.87 on 25 March 2012 - 02:51 AM in PCR, RT-PCR and Real-Time PCR

Than you for your reply!!

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 hope I've been clear... thanks again!!



#131686 Help with primer design

Posted by borriello.87 on 24 March 2012 - 05:35 PM in PCR, RT-PCR and Real-Time PCR

Hi all,

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?

Thanks in advance!!



#126303 Littermate control mice

Posted by borriello.87 on 02 January 2012 - 02:16 PM in Animal and Zoology

Hi all,

colud anyone explain me what "Littermate control mice" are?

I've never worked with mice...

Thanks in advance!!



#111141 Mitomycin C treatment to prepare feeder cells

Posted by borriello.87 on 27 May 2011 - 03:30 PM in Immunology

Hi all,
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 37C 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,

Francesco




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