primers for fungal identification - (Jan/20/2011 )

Hello,

Iam working on PCR based fungal identification.I had tried pimer sets ITS1-ITS4,ITS1F-ITS4B but there is no amplification.

Any suggestions? Thanks is advance.

Tried gradient PCR? can you provide more information regarding your mastermix and DNA preparation method before we troubleshoot?
Usually ITS primers set very less likely to fail... from my experience.

Yes you are right.ITS primers have always worked so far.

This time however maybe i need to try with other primer sets which i come across in literature searches,primers other than ITS1-ITS4 that i need to use.

DNA extaction was done from overnight grown cultures using our inhouse kit(frankly iam yet to find out the Solution A,B,C components).Annealing temperature is the standardized 55 degree Celsius for 1 minute.Haven't tried gradient so far.

Do you suggest direct scraping from plate and performing the extaction?Does that make any difference?And is there any protocol that should help? Thanks.

adrian kohsf on Thu Jan 20 21:55:16 2011 said:

Yes you are right.ITS primers have always worked so far.

This time however maybe i need to try with other primer sets which i come across in literature searches,primers other than ITS1-ITS4 that i need to use.

DNA extaction was done from overnight grown cultures using our inhouse kit(frankly iam yet to find out the Solution A,B,C components).Annealing temperature is the standardized 55 degree Celsius for 1 minute.Haven't tried gradient so far.

Do you suggest direct scraping from plate and performing the extaction?Does that make any difference?And is there any protocol that should help? Thanks.

I suspect is your DNA extraction problem.

Is your inhouse kit contain mechanical breakage of fungal cell wall, such as using mortar and pestle?

Try to use a conventional method which involved liquid nitrogen frozen, grinding with mortar & pestle and extract with phenol chloroform isoamyl alcohol..

Direct scraping less likely to work, from my experience, with exception of using yeast.

What is your fungal species? is it mold or yeast?

adrian kohsf on Sat Jan 22 18:33:53 2011 said:

Our kit does not contain the conventional method involving mortar-pestle.Maybe i should try the same.

Fungal species is mould.will re extracting this week and let you know the outcome.

Thanks once again,

Just a few notes:

I used to work with Aspergillus niger (AN) and Aspergillus terreus (AT). AT just need to boil with the lysis buffer and you can get the genomic DNA quite easy. But for AN, got no choice but to freeze in liquid nitrogen and grind it to fine powder before boil in lysis buffer.

Perhaps you just add in the "grind" step before proceed with your kit will do. Use 2cm x 1cm fresh mycellium (without spores).
BTW, convenient to tell which kit you use?

Adrian

You might also be having problems with inhibitors. I would suggest testing PCR with 10x and 100x dilutions of your DNA.

phage434 on Tue Jan 25 03:17:44 2011 said:

Wow, phage434, yes you are right and I do agree with you that dilutions are necessary...

adrian kohsf on Tue Jan 25 17:46:19 2011 said:

Hi!

i have met with success with one of the samples using all the suggestions i got here.should be through with the other two as well hopefully...

thanx alot:)