Pipettes and Pipetting

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Hello All,

At the risk of insulting your intelligence I am posting some basic information about pipettes and pipetting so you won’t have any excuses for poor practice at the lab bench. The main reason that it is important to use our pipettes correctly is that we share them. Its not just your work that will be affected if you seriously contaminate a pipette or throw it out of calibration.

If you have an undergrad or new volunteer or employee doing anything with our pipettes show them this post and make them read the manual (below).

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Drooping flowers cured?

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Earlier this year, Kate’s sunflowers in the hort greenhouse were having an issue where the stem right under a maturing flower would darken and wither. The flower would droop down and gradually die. This was particularly annoying when trying to collect seed from crosses.

I’ve been growing H. bolanderi in the hort greenhouse and my plants seemed to have the same issue. When I saw this happening I trimmed off the drooping flowers, which occurred on about 10 of 24 plants. I also noticed that the new leaves were browning at the tip, a symptom of nutrient burn, so I flushed the soil by excessively watering it once.

A week later my plants no longer had any head droop and some flowers that seemed iffy before were fine. The leaf tip browning also reduced. I’m not sure if flushing the soil was the cure or something else happened at the greenhouse or it has only temporarily subsided. This is just something to try if you have the same problem.

One interesting note is that my plants are serpentine adapted and Kate’s are dune plants, which are both fairly nutrient poor soils. Perhaps they are extra susceptible to nutrient burn.

lab contact information and birthday list

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Here is the most up-to-date contact information for the Rieseberg lab, including birthdays, email addresses, and phone numbers. If you want, you can download a word document of this here: Lab Contact Info and Birthday List. If your information changes, either let me know or edit this post and the downloadable document appropriately. Thanks!
(contact: John L)

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Rosalind: Learn Bioinformatics Online Through Problem Solving

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I was introduced to Rosalind, a problem-based Bioinformatics Tutorial website, and I think it’s fantastic.  I wish I knew about it when I was first starting out.  You can solve the problems in any language you want.  The website does not run your code.  It only grades your solution dataset.  There are a large number of problems on different topics, from codon-finding to protein spectrum matching.  I would say the problems are geared towards the beginner, but there is enough variety that a higher level bioinformaticist would also benefit from rounding out their knowledge.

Room Temperature RNA extraction

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Derivation: The following protocol is a modified Zhao Lai et al 2006 and TRizol Plus protocol.

Expected results: I tested the protocol with Arabidopsis seedlings and it gives quality RNA that was used for RT-PCR (non-quantitative).  I am able to achieve 75-270 ng/uL (100 uL elution – take this value with a grain of salt since it is derived from the nanodrop) of RNA from 38-48 mg of seedlings (this is approximately 20 seedlings grown for 18 days). I have yet to experience a failed RT-PCR with RNA isolated using this extraction.

Keys to success: Yield is highly linked to tissue mass and quality of tissue grind. The range of tissue mass is very small. One should use a small tissue amount and process a given sample over a column many times. Tissue is ground successfully only when it is not visible in the trizol/tissue/bead mixture. Minimize RNAse paranoia – Guanidine isothiocyanate does a good job of destroying protein structure, inactivating RNAses.

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GBS protocol Version 2.0

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Hi all,

Here is the long awaited updated GBS protocol.

PROTOCOL ->>>> GBSv2.0

There are three main changes from the previous protocol.

-After digestion and ligation, all the product is kept so more attempts can be made at the PCR.

-The PCR uses Phusion Taq, has longer extension times and one additional cycle with more primers/Taq.

-Size selection is done using AMPure beads instead of gel extraction.

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Plastid genome assembly from low coverage WGSS data

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This post describes the steps I took to assemble plastid genomes from low-coverage WGSS data. An overview of the approach can be found here.

Essentially, the method involves first mapping of quality-filtered reads to a reference plastid genome, and only selecting plastome-like reads from this mapping step for subsequent de-novo assembly. For the assembly step, I used the VELVET assembler, which performs well for small genomes and is quite fast.

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GBS multiplexing

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GBS_mutliplexing contains 2 scripts that may be useful to you if you are using GBS data. One essentially formats GBS reads for Tassel. The other demultiplexes the reads. A readme file in there explains things in more detail.

Edited: edited the readme and added a script to convert qseq to fastq

Seed size in H. exilis

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Here is a curiosity I noticed while sorting seeds.

At one of my H. exilis sites (G136) there was a serpentine field beside a raised road. The field had a large population of tiny sunflowers, but there were also a few plants on the gravel embankment beside the road. These plants were much bigger, probably because the soil used in the embankment wasn’t serpentine. I collected seeds from both field and roadside plants.

The roadside seeds are much bigger than the serpentine seeds.

This has several possible explanations:

-Plasticity. The non-serpentine plants are much bigger, being bigger makes their seeds also larger.

-Selection. Bigger seeds are better on the disturbed habitat of the gravel embankment.

-Introgression. Gene flow from H. annuus could be coming in and only persisting on non-serpentine areas, bringing larger seed alleles.

Say hello to my little friends

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It is my pleasure to introduce a new species to the Rieseberg lab: Alliaria petiolata (aka garlic mustard) is an invasive species in North America that is also widespread in Europe. Unfortunately we have had some problems with fungus and getting seeds to germinate, but we are moving forward.

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Germplasm pedigree

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This is a pretty cool figure that is hard to find, I got a hard copy sent to me and scanned it. It is a pedigree of all the publically available lines of sunflower up until 1989. I plan on cleaning this image up and possibly scanning it again, I thought I would save it on here in the mean time. It looks okay if you zoom in.

It is from this paper:

Korell, M., Mosges, G., and Friedt, W., (1992) Construction of a sunflower pedigree map. Helia 15: 7-16

Helianthus neglectus collecting trip – Oct 2012 (Kate)

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Recently, Kieran and I travelled to Monahans, Texas to collect Helianthus neglectus. It was a quick, fun trip in which we collected 8 dune and 10 non-dune populations. The seeds we collected are sorted and available in the lab in two boxes labelled “Helianthus neglectus – Monahans, Texas – Oct 2012″. Finally, I’ve posted the GPS coordinates and (usually) two photos for each site we collected from below. I will add more information about the habitat characteristics (vegetation cover and soil components) as it becomes available.

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Obscure Sunflower CMS References

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Hello,

I recently took advantage of Hannes, our man in the FAO, to obtain two obscure references about CMS in cultivated sunflower. Hannes, or rather his intern Brian, has sent me scans:

Serieys (1996) Identification, Study And Utilisation In Breeding Programs Of New CMS Sources. Helia 19:144-160.

Serieys (????) Identification, Study And Utilisation In Breeding Programs Of New CMS Sources. FAO Progress Report 1999-2001.

If you have any interest in the cms literature you have probably noticed that everybody cites these papers to make the claim that there are many sources of cms in sunflower. Now you can too.

Thanks Hannes, and thanks Brian.

 

Dan.

 

Blast2GO

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This describes how you can run blast2go on a server using b2gpipe and a local database. This makes blast2go a viable option for annotating large fasta files. Otherwise it is much too slow. The database is currently set up on an AdapTree server. This took a while for me to troubleshoot, so you could run into different problems, but you will hopefully avoid some of the issues I ran into. The b2g Google group is good for troubleshooting. You can find many of these instructions at http://www.blast2go.com/b2glaunch/resources/35-localb2gdb

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Allowed File Types At RLR – you can now upload scripts with their usual file extensions

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Hello All,

Many of us have been annoyed by the restricted file types that WordPress allows to be uploaded to RLR. It’s especially annoying because all WordPress is doing when it permits or denies an upload is checking the file extension against a list of allowable extensions. {Even the most malicious code could be uploaded to our blog as long as it had a .txt file extension. Whether that code could then be made to execute, however, is far beyond my web-programming grasp – WordPress would treat it as plain text so it may be impossible.}

We’ve been sharing code via RLR by sidestepping the file extension rules and uploading scripts as .txt text files or by compressing files into zip archives or just putting the code itself into posts. Admittedly these were simple solutions, but now it’s even simpler – I just added some of the relevant file extensions to the list that RLR will allow for upload.

I added: “.pl”, “.py”, “.sh”, “.R”, “.r” and “.kml”.

Any file with one of those extensions will upload as plain text, i.e. WordPress will treat it as a text file.

If I’ve omitted something useful let me know.

Please remember that code can simply be copied into the body of a post and that will often be the best way to share it. But, in addition to that presentation, and especially for long scripts, you can now upload the script with its file extension to the RLR media library and put a link to it in your helpful post explaining what it does.

Dan.

Newest Lab Member – Needs Name

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Dear Colleagues,

It is my pleasure to introduce you to our newest lab member… actually it doesn’t have a name yet, but that’s the main reason for this post. This cute little guy/girl needs a name. I have been toying with TERMINATOR, HAL, iRob, and Rob(ot), but I’m sure you can do better. Could be male, female, or gender-neutral and whoever comes up with the best name gets a special treat.

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