Pipettes and Pipetting

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).

Continue reading

Obscure Sunflower CMS References


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.




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

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.


Text File To kml – Perl Script

Google Earth reads and writes a special form of xml file called a kml (keyhole markup language). Many other geographic viewers and GISs can also read kml files so it’s not a bad thing to be able to make kml files for sample location data. I assume there are many ways to do this. The way I have done it is via a perlscript that I wrote. This post provides that script and explains what it does.

Here is the script, its called texttokml.pl.

It’s very simple and I commented it heavily so even the most naive perl programmer should be able to figure it out and change it but if you want me to hold your hand just ask.

Explanation follows . . .

Continue reading

RLR Image Library (Dan E.)

Hello all. I’ve created an image library here at RLR. I think its a good idea and I’m hoping that you do too. If we accumulate a collection of good images, mostly photos I assume, it will become useful and interesting.

The idea is that we can post image galleries – collections of photos of or about a particular project/trip/experiment/event – to share with the lab. I hope this sharing will be informative and entertaining but also practical – we can share images for use in presentations and posters and the like.

Right off the bat I want it to be clear that if you use any image from RLR that you did not create yourself, that image must be attributed to its creator. Its easy, just give the person who made and uploaded the image a credit on or near the image in your presentation or poster etc.. Pretty obvious really.

The image library comprises galleries displayed on new pages added to RLR under the “Image Library” page. If you wish, these pages can be hidden such that only registered users who have logged in can see your images.

Check out Brook’s opening effort for an excellent example of what I’m talking about.

Greg O. has also put up some nice shots of Californian sunflowers.

I’ve added instructions on the “How to: contribute content” page and on the “How to: use RLR” page.

If my instructions are insufficient, or you can see obvious problems or improvements, please let me know.

Answers to some questions that you may have . . .

Continue reading

Making Illumina Whole Genome Shotgun Sequencing Libraries – (Dan E.)

I’ve been making whole genome shotgun sequencing libraries (for the purposes of this post: WGSS libraries) to sequence sunflower genomes on the Biodiversity Centre’s Illumina HiSeq. I haven’t been doing it for very long and its likely that my approach will change in the future as costs and products change but, as of early 2012, I’ve landed on a hybrid protocol based on kits from an outfit called Bioo Scientific. I use the Bioo Sci. adapter kit and their library prep kit up to the final PCR step at which point I switch to a PCR kit from another outfit called KAPA. I also use a KAPA kit to quant libraries with qPCR. In this post I give a little context then describe what I do to make WGSS libraries . . . Continue reading

Illumina Sequencing Adapters and Barcodes (Dan E.)

As of March 2012 we are using the Bioo Scientific NEXTflex barcoded adapters for WGS sequencing libraries made by ourselves, (well me so far). The set we are currently using comprises 48 barcodes, so we can multiplex up to a 48-plex in one lane on the Illumina HiSeq sequencer.

Bioo Sci. 48 barcoded adapters

Below are the sequences of the Illumina adapters and the 48 barcodes we are currently using. Continue reading

Ampure XP beads – DNA clean up and size selection (Dan E.)

Ampure XP magnetic beads have become popular for cleaning DNA from reactions and for size selecting DNA. WGS sequencing library preparation protocols, and the GBS protocol, involve one or more bead clean up steps. If you make next gen sequencing libraries you will use Ampure XP beads. Following is some basic information about them and my thoughts about using them, mainly for library preparations . . . Continue reading

Loading Dye for Agarose Gels (Dan E.)

If you are new to the lab bench, and perhaps even if you’re not, you may not know how to make loading dye for agarose gels. Its the sort of thing that tends to get made rarely and then persist, so people often inherit a supply and have no idea how to make more when they run out. For this sort of thing the best reference is the relevant appendix at the back of the Sambrook et al. Molecular Cloning text books (near Megan’s desk) and, of course, there is Google. Or, you can just do what I do. My recipe is incredibly simple and would be close to the cheapest option too. Continue reading

Gel Slice – size selection or band isolation (Dan E.)

Cutting a slice from an agarose gel in order to size select DNA or to isolate a PCR product etc. is standard practice in lab genetics. This post is about how I do it in our lab, (as of Feb 2012).

There are three features of my current method that may be unfamiliar to you even if you’ve done this sort of thing before:

  1. The Dark Reader transilluminator.
  2. SYBR-Gold.
  3. Sigma X-Tracta gel punches.

Continue reading

Shearing DNA for WGS Sequencing- Bioruptor (Dan E.)

This post is about fragmenting, or shearing, genomic DNA to a particular size range using the Rieseberg lab’s Bioruptor sonicator.

Most of the current whole genome shotgun (WGS) library preparation protocols for NGS applications start with fragmented DNA. Generally speaking, this starting DNA should be a certain size and, for multiple samples, consistently that size. This objective turns out to be quite a tricky thing to accomplish with the Bioruptor. Given that WGS sequencing will probably continue to be popular in the lab, I am posting here what I have learned so far about taking whole genomic sunflower DNA and smashing it to the size range that I want using the Bioruptor. If I discover anything else in future library preps I’ll add it below. If anybody else has useful tips please comment.

Continue reading

How to post – code (Dan E.)

We have a problem sharing code via RLR.

The Problem

Unfortunately WordPress has a list of acceptable file types that it allows to be uploaded to our media library and none of the useful coding file types are on that list. The list is simply a list of acceptable file extensions. This means if you write a useful R script (or perl or python) script and save it with a standard file extension, like .R or .pl, WordPress will not allow you to upload it to the RLR media library so that you can share it via a post.

The Solution

The list of acceptable file extensions can be hacked and I might give it a try but, until I do, you will have to do one of these things:

  • Change the file extension. If you save your script as a .txt file it will upload fine. You should make it clear in your post what kind of script it is and then people who download it can change the .txt extension to whatever they want.
  • Put the code in your post. If your script is not too long you can simply copy and paste the code from your text editor into the post editor. The formatting of the code will remain true to the original so users can simply copy and paste it back out into a text editor or R-Studio or wherever. See Rose’s post about plotting STRUCTURE results for an example of this.
  • Compress your script file. If your script is big you can try zipping it and then uploading the compressed file. Users can then just download and unzip it. [As of November 2011 this hasn’t been tested.]

Dan E.

Collecting tissue for DNA (Dan E.)

Hello Rieseberglers.

Almost all of us collect tissue for DNA but there are always new people in the lab, some of whom may have never worked with plants, or even DNA I suppose, and who might benefit from the experience of seasoned plant geneticists. This post contains my advice on collecting sunflower, or other plant, tissue for DNA work. If you have any tips, or alternatives please contribute in the comments.
Continue reading

How to post- Continue reading-> (Dan E.)

Hi All.

Greg B has shown me a very useful HTML trick that will make the RLR Home page more user friendly if we all use it.

If you are just looking for the code so you can copy it, here it is:

UPDATE (Jan2012): I just noticed that there are two other, easier, ways to get the “Continue reading →” feature into your post.

  1. Keyboard shortcut: alt+shift+t.
  2. WordPress post editor button: There is an “insert more tag” button among the buttons at the top of the post editor. In my editor it is on the top row 4th from the right – beside the link and unlink buttons.

It is very simple to post to RLR such that your post is displayed on the Home page as an opening paragraph or two followed by a “Continue reading →” link that takes the reader to the full post when followed.

Just like this . . .
Continue reading

Quantifying DNA (Dan E.)

This post is about quantifying DNA samples in the Rieseberg lab, i.e. using the tools we have available to us in our lab in the Biodiversity building as of November 2011.

There are three ways to quantify the amount of DNA in an aqueous solution (e.g. DNA dissolved in water or TE):

  1. Nanodrop (spectrophotometry).
  2. Qubit (flourometry).
  3. Agarose gel with EtBr (also fluorometry -> UV lightbox + your eyes = fluorometer).
  4. (There is also the BioAnalyzer – I’m going to ignore that here and focus on methods to measure high mw genomic DNA).

These methods have different benefits and limitations but they are all valid and useful.
Continue reading