12 replies to this topic

[pihoo]

I m working over different fungus strains after PCR amplification with ITS1 and ITS4 primer that is universal primer.I got multiple bands ..i have query  abt that. Am i going to right direction or not?.... cuz in many of research papaer i found the single band there..n  so i m confused so please if anyone know about this exactly please leave the answer!

[Adrian K]

Try use ITS1F primer rather than ITS1, and see if there is any improvement. Did you optimize and run a gradient PCR to optimize?

[gebirgsziege]

from pure cultures ITS1/4 should usually produce a single band from pure cultures. I never experienced any superiority of ITS1F - and I have done a lot of fungal PCRs.

I experienced this problem once....doing 40 cycles instead of 25 or 30 solved the problem in my case (but I have no rational explanation for this besides that the demons of PCR required a longer time to be sacrificed to them - so if anybody has one I would appreciate to hear it .

I would also suggest you check your cultures for purity with the microscope (also look for Bacteria, yeast cells etc.).

is your extraction and PCR negative control ok, or do you have bands in it as well - there is the possibility of a contamination of your reagents.

as already suggested, gradient PCR might help; with most fungi you can go as high as 58/60 °C using ITS4/5, this will improve specificity and solve your problem.

Edited by gebirgsziege, 10 March 2011 - 09:00 AM.

[Maddie]

gebirgsziege, on 10 March 2011 - 09:00 AM, said:

from pure cultures ITS1/4 should usually produce a single band from pure cultures. I never experienced any superiority of ITS1F - and I have done a lot of fungal PCRs.

I experienced this problem once....doing 40 cycles instead of 25 or 30 solved the problem in my case (but I have no rational explanation for this besides that the demons of PCR required a longer time to be sacrificed to them - so if anybody has one I would appreciate to hear it .

I would also suggest you check your cultures for purity with the microscope (also look for Bacteria, yeast cells etc.).

is your extraction and PCR negative control ok, or do you have bands in it as well - there is the possibility of a contamination of your reagents.

as already suggested, gradient PCR might help; with most fungi you can go as high as 58/60 °C using ITS4/5, this will improve specificity and solve your problem.

Gebirgsziege, how long is the amplicon with ITS4 and 5?

[gebirgsziege]

not very long 600 bp (+/- 100 bp). procession times we use are quite short:  annealing & denaturation 15s, elongation 40s.

Do you have an idea what might have happened with my sample back then?

[pihoo]

Yes! i have standardize the  the annealing temperature according to their melting temperature in gradient PCR..it was bteween 58 to 62..n i got at every temp multiple bands . some of strain which i took was pure form..i mean were no contamination there..at that time.. if during PCR or making of master mixture ..it might be contaiminate...well thanks for the response

[pihoo]

adrian kohsf, on 10 March 2011 - 08:41 AM, said:

Try use ITS1F primer rather than ITS1, and see if there is any improvement. Did you optimize and run a gradient PCR to optimize?

yes i have optimized and set my annealing temp according to the primer melting temperature!

[Adrian K]

I agree with gebirgsziege, you should check your extraction and PCR negative control. Although I didn't so as much fungal PCR as gebirgsziege did, but i never experience multiple band issue with my ITS primers set yet.

p/s: Briefly, based on the morphology, is it a yeast, mold or mushroom? which fungus you "suspect" it might be? any idea? Is your template preparation room and PCR room clean?

[pihoo]

adrian kohsf, on 11 March 2011 - 10:30 AM, said:

I agree with gebirgsziege, you should check your extraction and PCR negative control. Although I didn't so as much fungal PCR as gebirgsziege did, but i never experience multiple band issue with my ITS primers set yet.

p/s: Briefly, based on the morphology, is it a yeast, mold or mushroom? which fungus you "suspect" it might be? any idea? Is your template preparation room and PCR room clean?

i just wann know that fungal amplification amplified with ITS1i.e forward primer and ITS4 i.e reverse primer produces the single bands..thats it only...actually i was confused between multiple and single band..cuz i have never worked on it before .thats why!...i have fungus pure strain ..with their name..so i also think that it should produce single band cuz u see ITS i.e internal transcribed spacer are the specific sequences present between 18s rRNA , 5.8 S rRNA . and 5.8 S rRNA AND 28 S rRNA ..so on every DNA molecula of same individual have the same size ITS sequence ON their respective rRNA GENE.right! So it must produce same base pair of bands have same mol.wight.AM i going right???? or not ??? PLz VERIFY IT.

[pihoo]

gebirgsziege, on 10 March 2011 - 09:00 AM, said:

from pure cultures ITS1/4 should usually produce a single band from pure cultures. I never experienced any superiority of ITS1F - and I have done a lot of fungal PCRs.

I experienced this problem once....doing 40 cycles instead of 25 or 30 solved the problem in my case (but I have no rational explanation for this besides that the demons of PCR required a longer time to be sacrificed to them - so if anybody has one I would appreciate to hear it .

I would also suggest you check your cultures for purity with the microscope (also look for Bacteria, yeast cells etc.).

is your extraction and PCR negative control ok, or do you have bands in it as well - there is the possibility of a contamination of your reagents.

as already suggested, gradient PCR might help; with most fungi you can go as high as 58/60 °C using ITS4/5, this will improve specificity and solve your problem.

Thnks alot but one more favor i wann just plz clear me out that pure fungus strain( having name) produces the single bands.i just wann know that fungal amplification amplified with ITS1i.e forward primer and ITS4 i.e reverse primer produces the single bands..thats it only...actually i was confused between multiple and single band..cuz i have never worked on it before .thats why!...i have fungus pure strain ..with their name..so i also think that it should produce single band cuz u see ITS i.e internal transcribed spacer are the specific sequences present between 18s rRNA , 5.8 S rRNA . and 5.8 S rRNA AND 28 S rRNA ..so on every DNA molecula of same individual have the same size ITS sequence ON their respective rRNA GENE.right! So it must produce same base pair of bands have same mol.wight.AM i going right???? or not ??? PLz VERIFY IT.

[pihoo]

gebirgsziege, on 10 March 2011 - 11:52 PM, said:

not very long 600 bp (+/- 100 bp). procession times we use are quite short:  annealing & denaturation 15s, elongation 40s.

Do you have an idea what might have happened with my sample back then?

one more query i have plz clear it if possible ....amplification with ITS1 n produces the bands of size generally between 500 to 700 bp...and amplification with ITS4 also produces bands of size between 500 to 700 bp then why we work with two primer.is one primer is not enough to make differentiation between genus..Plz reply..means there slightly difference between  two different strain( different genus)..of base pairs.so how can i make diffentiation between them..n how dendrogram will be drawn?

[Adrian K]

pihoo, on 12 March 2011 - 08:17 AM, said:

i just wann know that fungal amplification amplified with ITS1i.e forward primer and ITS4 i.e reverse primer produces the single bands..thats it only...actually i was confused between multiple and single band..cuz i have never worked on it before .thats why!...i have fungus pure strain ..with their name..so i also think that it should produce single band cuz u see ITS i.e internal transcribed spacer are the specific sequences present between 18s rRNA , 5.8 S rRNA . and 5.8 S rRNA AND 28 S rRNA ..so on every DNA molecula of same individual have the same size ITS sequence ON their respective rRNA GENE.right! So it must produce same base pair of bands have same mol.wight.AM i going right???? or not ??? PLz VERIFY IT.

pihoo, on 12 March 2011 - 08:28 AM, said:

one more query i have plz clear it if possible ....amplification with ITS1 n produces the bands of size generally between 500 to 700 bp...and amplification with ITS4 also produces bands of size between 500 to 700 bp then why we work with two primer.is one primer is not enough to make differentiation between genus..Plz reply..means there slightly difference between  two different strain( different genus)..of base pairs.so how can i make diffentiation between them..n how dendrogram will be drawn?

I suppose you run ITS1 primers in 1 tube, and ITS4 in another tube, right? I hope you are not doing that.

Based on your question I can say that you had got things mixed up here. ITS1 and ITS4 means we using both primers together in a single amplification tube to get one band which is between 500 to 700 bp. We sequence the amplicon and identify the strains. They not necessary different by base pairs, they can be different by DNA compositions as well. You run your sequenced DNA through software like phylip or clustalX and let the software worry about your dendograms. Did I answer your question?

And if you have your protocol or gel picture you are always welcome to post it here so we can help you troubleshoot your problem.

Also, repeat posting the same question over the forum is not always the good idea. I understand there are frustrations in research but please bear with it as you are not the only one going through such process.

[pihoo]

adrian kohsf, on 13 March 2011 - 08:06 AM, said:

pihoo, on 12 March 2011 - 08:17 AM, said:

i just wann know that fungal amplification amplified with ITS1i.e forward primer and ITS4 i.e reverse primer produces the single bands..thats it only...actually i was confused between multiple and single band..cuz i have never worked on it before .thats why!...i have fungus pure strain ..with their name..so i also think that it should produce single band cuz u see ITS i.e internal transcribed spacer are the specific sequences present between 18s rRNA , 5.8 S rRNA . and 5.8 S rRNA AND 28 S rRNA ..so on every DNA molecula of same individual have the same size ITS sequence ON their respective rRNA GENE.right! So it must produce same base pair of bands have same mol.wight.AM i going right???? or not ??? PLz VERIFY IT.

pihoo, on 12 March 2011 - 08:28 AM, said:

one more query i have plz clear it if possible ....amplification with ITS1 n produces the bands of size generally between 500 to 700 bp...and amplification with ITS4 also produces bands of size between 500 to 700 bp then why we work with two primer.is one primer is not enough to make differentiation between genus..Plz reply..means there slightly difference between  two different strain( different genus)..of base pairs.so how can i make diffentiation between them..n how dendrogram will be drawn?

Thnks!

I suppose you run ITS1 primers in 1 tube, and ITS4 in another tube, right? I hope you are not doing that.

Based on your question I can say that you had got things mixed up here. ITS1 and ITS4 means we using both primers together in a single amplification tube to get one band which is between 500 to 700 bp. We sequence the amplicon and identify the strains. They not necessary different by base pairs, they can be different by DNA compositions as well. You run your sequenced DNA through software like phylip or clustalX and let the software worry about your dendograms. Did I answer your question?

And if you have your protocol or gel picture you are always welcome to post it here so we can help you troubleshoot your problem.

Also, repeat posting the same question over the forum is not always the good idea. I understand there are frustrations in research but please bear with it as you are not the only one going through such process.