Hey Ammar,

I guess you need it to check transformants? I work on Candida albicans and the following protocol works very well for us:

Pick a colony, disolve in 10 µl of ddH2O and boil for 5 minutes at 95°C in thermocycler.

Prepare a Master Mix with the following reagents per reaction:

+ 3.1 µl ddH2O

+ 2 µl 10x Taq buffer

+ 1.6 µl 2.5 µM dNTPs

+ 1.6 µl 2.5 µM forward primer

+ 1.6 µl 2.5 µM reverse primer

+ 0.125 µM Taq polymerase

Add 10 µl of the Master Mix to each boiled colony.

95°C - 2 min

-

95°C - 20 sec

52°C - 20 sec

68°C - 60 sec, 35 cycles

-

68°C - 5 min

Annealing temperature and elongation time depend of course on your primers and PCR product, respectively.

I hope that helps!

Best,

Philipp

You can get lots of protocols on this subject on protocol.oi from plos journal just create account and login.

try this

dx.doi.org/10.17504/protocols.io.gzwbx7e

Philipp Reuter-Weissenberger thank you a lot, it worked very well. I use it for transformation.

if you work with C.albicans, did you use FACS analysis ?

Yes, I did but I think our protocols do not really differ from FACS protocols working on other organisms. We have an Accuri Cytometer which adjusts the scatter automatically. I think it is a good idea to talk to the people in your institute who worked with the FACS machines that you have. We aim for 200 to 1000 events/second.

Best

Philipp

Ammar Abou Kandil The following article will be helpful for you. Have a look.

1. Lau, A., Sorrell, T. C., Lee, O., Stanley, K., & Halliday, C. (2008). Colony multiplex-tandem PCR for rapid, accurate identification of fungal cultures. Journal of clinical microbiology, 46(12), 4058-4060.