IF ITS1 and ITS4 using as primer and working on the fungi . Shall we get single - (Mar/10/2011 )
gebirgsziege on Fri Mar 11 07:52:47 2011 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?
-pihoo-
pihoo on Sat Mar 12 16:17:26 2011 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 Sat Mar 12 16:28:44 2011 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.
-adrian kohsf-
adrian kohsf on Sun Mar 13 16:06:44 2011 said:
pihoo on Sat Mar 12 16:17:26 2011 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 Sat Mar 12 16:28:44 2011 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.
-pihoo-