"Hi Conrad, It's great discussion. I suggest having the posibility to include different ITS copies found in the same thallus. There are some cases where different ITS can be found in the same thallus within the range of a single species ITS…"
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Pradeep Kumar Divakar
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Pradeep Kumar Divakar's Discussions
PCR protocol
1 Reply
Hi Conard,As per your suggestion, I am hereby adding a PCR protocol for MCM7 gene.Best primer combinations and annealing temperature:Forward primer: Mcm7-709for: ACIMGIGTITCVGAYGTHAARCCReverse…Continue
Started this discussion. Last reply by Kris Jett Nov 19, 2010.
Pradeep Kumar Divakar's Page
Latest Activity
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'ITS barcode requirements V2' in the group Fungi
Conrad Schoch replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Imke Schmitt recently sent me this updated protocol for MCM7. I am posting it here at her request for those who may want to give this gene another go as phylogenetic marker.
Tips for MCM7 amplification:
Use a sensitive polymerase, e.g.…"
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'Species and barcoding (Thorsten's proposed collaborative project)' in the group Fungi"Dear all,
This is a very nice idea and suggestion by Thorsten Lumbsch on species delimitation using GMYC method and good way to explore the datasets to have separate publication. Species delimitation is a crucial issue for DNA barcoding. Groups like…"
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'RPB1 and 2 problems' in the group Fungi"Apparently, for parmeliaceae now RPB1 is working good but we have to be very careful in primer sets selection, to be sure that the primer we are using are the original ones which is published in Stiller & Hall, 1997 and Matheny & al.,…"
Pradeep Kumar Divakar commented on Conrad Schoch's group 'Fungi'"Dear all,
This is a very nice idea and suggestion by Thorsten Lumbsch on species delimitation using GMYC method and good way to explore the datasets to have separate publication. Species delimitation is a crucial issue for DNA barcoding. Groups like…"
Conrad Schoch replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Thanks Ryan, any info you add will be helpful to many I am sure. Would be good to expand things beyond Ascos."
Ryan Kepler replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Hey everyone,
Looks as though most of the people commenting here are working with Ascomycota. Thought I would chime in with some successes for Basids. I am in the process of adapting these for use with Amylostereum in Russulales.…"
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'RPB1 and 2 problems' in the group Fungi"Hi Karen,
Thanks a lot for your kind reply and details of pcr protocol. Hope we would be able to resolve this "mistake".
Giho Sung could you please tell us the original citation of this primer…"
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'RPB1 and 2 problems' in the group Fungi"Hi Conrad,
I was revising the RPB1 primers listed in excel file (PrimersTable30November) because we are having some problem in sequencing of this loci. Primer name RPB1-Af, RPB1-Ac oligonucleotides: 5’-GARTGYCCDGGDCAYTTYGG-3’ I did not…"
Pradeep Kumar Divakar replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"We also use the same concentration for Tsr1 (1.25ul of 10mM). We did not try increasing more primer concentration but i think it should also work better by increasing primer concentration.
regards
divakar
"
Sandra Lorena Ament Velásquez replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"I see. Thanks a lot! The same applies for Tsr1, right?Regards,Lore"
Pradeep Kumar Divakar replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Hi Sandra,
1.25ul of each 10mM primer in 25ul PCR reaction is not much. We normally use this for amplification of all genes (nuclear, mitochondrial and protein coding) and have obtained very nice results. Recently we got good result for protein…"
Andrew Miller replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"We use 1.0µL of each 10mM primer in our 25µL PCR reactions.
Andy"
Sandra Lorena Ament Velásquez replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Hi everyone!I read the paper of Schmitt et al. (2009), but i have a doubt. It says that the concentration of the primers for de PCR is 10mM in 1.25uL. Is it really that much?Greetings!Lore"
Pradeep Kumar Divakar replied to Conrad Schoch's discussion 'RPB1 and 2 problems' in the group Fungi"Hi Conard,
In lichenized Ascomycota we wound good result in the amplification of RPB1 by increasing primer concentration. We used 3ul of each primers For and Rev (10uM) in 25ul of reaction. For RPB1, we are using universal primers RPB1-Af and…"
Conrad Schoch replied to Pradeep Kumar Divakar's discussion 'PCR protocol' in the group Fungi"Tried these on a diverse set of Dothids and I think success rate was more like 80% without too much optimisation."
Profile Information
- Institution/Organization
- Facultad de Farmacia, Universidad Complutense de Madrid, Spain
- Title/Position
- Researcher/Faculty member
- I’m involved in, or am interested in the following barcoding initiatives
- TreeBOL, DNA Barcoding Initiative for Conservation, Plant Working Group, Database Working Group, Data Analysis Working Group
- I’m involved in or interested in the following iBOL Working Groups
- WG1.2 Land Plants, WG1.3 Fungi, WG2.3 Methodological Innovation
- Keywords about my barcoding projects (separate each choice with a comma)
- Fungi, Lichens, Ascomycetes
- I’m involved in the following aspects of barcoding:
- Fieldwork/specimen collecting, Labwork/generating barcode sequences, Management of barcode data, Analysis of barcode data, Using barcode data in taxonomy/systematic biology
- Current Project(s)
- Developing species DNA barcoding in lichen forming fungi using the Parmeliaceae as model. Funded by Ministry of Science and Innovation, Spain.
Pradeep Kumar Divakar's Photos
Comment Wall (1 comment)
- At 9:12am on October 4, 2010,
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