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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,


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