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the pyrithiamine


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5 replies to this topic

#1 Gaida

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Posted 28 July 2010 - 05:37 AM

Hallo all,

I have problem with the pyrithiamine concentration,
I tried to use concentrations from 0.1ug /ml up to 1ug/ml of pyrithiamine in Czapek-Dox(CD) medium with Ph6.5 at 37C for Aspergillus fumigatus to select transformed colonies (Aspergillus fumigatus)

but unfortunately, I got non transformed colonies were grown in selective( pyrithiamine)medium

What can I do?

Thanks

#2 perneseblue

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Posted 28 July 2010 - 05:19 PM

How about doing ai titrated kill curve?
Find out what concentration of pyrithiamine you need to get a 100% kill rate.
May your PCR products be long, your protocols short and your boss on holiday

#3 Gaida

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Posted 29 July 2010 - 04:17 AM

0.1 ug/ml is the concentration to kill aspergillus fumigatus

but i used 1ug/ml and still aspergillus fumigatus grow

#4 perneseblue

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Posted 29 July 2010 - 09:17 PM

0.1 ug/ml is the concentration to kill aspergillus fumigatus

but i used 1ug/ml and still aspergillus fumigatus grow


How about using a higher concentration?

Was a titrated kill curve conducted?

And, might the false positives be contamination?

Alternatively might the transformation have failed. Thus you are culturing the plate to the point where the selection agent breaks down allowing for nontransformed colonies of fungi to grow.
May your PCR products be long, your protocols short and your boss on holiday

#5 Gaida

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Posted 02 August 2010 - 05:05 AM


0.1 ug/ml is the concentration to kill aspergillus fumigatus

but i used 1ug/ml and still aspergillus fumigatus grow


How about using a higher concentration?

Was a titrated kill curve conducted?

And, might the false positives be contamination?

Alternatively might the transformation have failed. Thus you are culturing the plate to the point where the selection agent breaks down allowing for nontransformed colonies of fungi to grow.



#6 Gaida

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Posted 02 August 2010 - 06:29 AM


0.1 ug/ml is the concentration to kill aspergillus fumigatus

but i used 1ug/ml and still aspergillus fumigatus grow


How about using a higher concentration?

Was a titrated kill curve conducted?

And, might the false positives be contamination?

Alternatively might the transformation have failed. Thus you are culturing the plate to the point where the selection agent breaks down allowing for nontransformed colonies of fungi to grow.


Thank you for you advices
Inspire of all paper I read, they used .1mg/l, I tried to increase the concentration of Pyrithiamin and now still in the incubation 37C


I do not know when agent breaks down?
So may be Aspergillus after this point can grow




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