5 replies to this topic

[Gaida]

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

[perneseblue]

How about doing ai titrated kill curve?  
Find out what concentration of pyrithiamine you need to get a 100% kill rate.

[Gaida]

0.1 ug/ml is the  concentration to kill aspergillus fumigatus

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

[perneseblue]

Gaida, on 29 July 2010 - 04:17 AM, said:

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.

[Gaida]

perneseblue, on 29 July 2010 - 09:17 PM, said:

Gaida, on 29 July 2010 - 04:17 AM, said:

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.

[Gaida]

perneseblue, on 29 July 2010 - 09:17 PM, said:

Gaida, on 29 July 2010 - 04:17 AM, said:

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