IF ITS1 and ITS4 using as primer and working on the fungi . Shall we get single - (Mar/10/2011 )
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!
Try use ITS1F primer rather than ITS1, and see if there is any improvement. Did you optimize and run a gradient PCR to optimize?
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 on Thu Mar 10 17:00:20 2011 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?
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?
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
adrian kohsf on Thu Mar 10 16:41:39 2011 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!
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?
adrian kohsf on Fri Mar 11 18:30:33 2011 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.
gebirgsziege on Thu Mar 10 17:00:20 2011 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.