"Hi Becky,
Nice to hear from you too...! I tried to modify my answer but apparently it did not work. I wanted to precise that even with dilutions or modifying the PCR program you will not avoid lichen contaminants. This problem is…"
international online community for dna barcoding professionals
Rebecca Yahr's Page
Latest Activity
Rebecca Yahr replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Hi Val -
Nice to hear from you! Yes, I recall lots of cloning in V Reeb's lichen LSU life and I have no doubt there are lots of goodies living inside as well. THere are some interesting ideas floating around, so I will see where I can get with…"
Rebecca Yahr replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Thanks Isaac - An interesting idea and not the first time I've heard it. I will try it with a few of the recalcitrant ones!
"
Rebecca Yahr replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Thanks Karen. I will try some other primers for sequencing."
Valerie Hofstetter replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Me again...
I also tried to avoid contaminant amplification. The major problem is that lichenized fungi have often many more introns than the non-lichenized one and the PCR is biased toward the shorter fragment. With much more template DNA of…"
Valerie Hofstetter replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Hello Rebecca,
Working on Usnea, I bet you have multiple bands because of lichen endophytes. I always had that problem with ribosomal genes of lichens in the AFTOL project. I am afraid that cloning will be the only solution...
All the…"
Isaac Chow replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Hi Rebecca,
I have experienced same band size with multiple amplicons, except cloning, I have figured out a easier method to solve this, keep everything but except doing 1:10 dilution of the DNA template, do a 1:2 and so on, ignore the…"
"Hi Rebecca.
If you really do not want to clone, there are several alternative methods to optimize your PCR and reduce your bands.
1) Use less DNA template in your PCR reaction, adding to much DNA could be messing up…"
Rebecca Yahr replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Hi William,
I'm working on Usnea in the Lecanoromycetes. I have had some advice about PCR protocols, but even with the much higher annealing temperature I'm using, I'm still getting the multiple bands for some samples. Sequencing of…"
"Hi Rebecca.
Could you please tell us what species/genera you are working on?
The optimal method of amplifying loci via PCR can vary per genera, also something like as your PCR mix can matter.
If you can provide us with…"
Karen W. Hughes replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Hi Rebecca,
We have this problem from time to time and it's been due to intron presence of variable sizes. Usually, the introns are adjacent to the LR5 primer. To avoid excising PCR bands or cloning, you could try sequencing from the left…"
María P. Martín replied to Rebecca Yahr's discussion 'LSU - multiple bands' in the group Fungi"Dear Rebecca,
If you have multiple bands with high concentration of DNA, it would be good at the beginning of your study to run a electrophoresis gel, cut every band and purify separately (even though that you comment that you…"
Rebecca Yahr added a discussion to the group Fungi
LSU - multiple bands
Dear All, I am new to sequencing LSU, and I find I have multiple bands on my PCRs (using LR5 and LROR with a two-stage PCR: 5 cycles annealing 61.5deg and 30 cylces at 59). Some come out with single lovely and sequencable bands of about 1000bp; some have two or three bands with 1300 or closer to 1800bp. The longest band is invariably nice and bright. I am expecting to find group I intron insertions to explain these extra bands (will be sequencing them shortly).I would like to avoid…See More
Rebecca Yahr joined Conrad Schoch's groupFungi
To provide up-to-date information on fungal barcoding and to facilitate communication and the development of collaboration.
Profile Information
- Institution/Organization
- Royal Botanic Garden Edinburgh
- Title/Position
- Researcher/Faculty member
- If Other Title, please specify
- Lichen Biodiversity Scientist
- I’m involved in or interested in the following iBOL Working Groups
- WG1.3 Fungi, WG2.1 Barcoding Biotas
- Keywords about my barcoding projects (separate each choice with a comma)
- lichen, usnea, cladonia
- I’m involved in the following aspects of barcoding:
- Fieldwork/specimen collecting, Museum work/specimen curation, Labwork/generating barcode sequences, Management of barcode data, Analysis of barcode data, Using barcode data in taxonomy/systematic biology
Comment Wall (1 comment)
- At 10:55am on October 20, 2010,
Matthew Sileo said… - Hello and welcome!
Thank you for taking the time to join. Here are a few things that you can do in the community.
After completing your online profile, please add yourself to the member map. Next, invite your colleagues and search our members for people you know and invite them to be your colleague in the network. Or browse our Forum and ask a question.
Please join a Group or start your own, as groups are a great way to express your special interest, be it a taxonomic group, a regional or organizational affiliation.
Have a question? Ask one of our hosts.
A few more...
Please contribute to our Blog. Simply choose “Add a Blog Post” under Blogs.
If you already have a blog, you can share it through RSS either on your page or on our rss pages. Just ask me how.
Or create a group and invite colleagues to join. Use “Send Message to Group” to email everyone in your group, start discussions specific to your group’s interests, add widgets and more.
For assistance, don’t hesitate to contact me.
Again, thank you for joining our growing online community. I look forward to connecting with you soon.
-Matt Sileo
© 2012 Created by Matthew Fisher.
Powered by
