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primers for fungal identification


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9 replies to this topic

#1 mehere

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Posted 20 January 2011 - 01:34 AM

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.

#2 Adrian K

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Posted 20 January 2011 - 01:55 PM

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.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 mehere

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Posted 21 January 2011 - 04:11 AM

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.

#4 mehere

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Posted 21 January 2011 - 04:25 AM

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.

#5 Adrian K

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Posted 22 January 2011 - 10:33 AM

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?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#6 mehere

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Posted 23 January 2011 - 11:08 PM

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?




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,

#7 Adrian K

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Posted 24 January 2011 - 07:13 AM

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
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#8 phage434

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Posted 24 January 2011 - 07:17 PM

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

#9 Adrian K

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Posted 25 January 2011 - 09:46 AM

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


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

Edited by adrian kohsf, 25 January 2011 - 09:48 AM.

Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#10 mehere

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Posted 15 February 2011 - 09:47 PM


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


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




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:)




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