Bioanalyzer – RNA (Kristin)

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Running a RNA Nano chip is very similar to running a DNA chip on the Bioanalyzer. However there are some things to keep in mind:

1. Clean everything with RNAse Away! before you start. I clean the chip loading stand as well as everything I am working with.

2. Make sure the reagents are at room temperature before you start to make the gel.

3. Before loading the chip, pipette 1.2 uL of each sample as well as the ladder into PCR tubes and incubate in the thermocycler for 2 minutes.

On the last point, I aliquot my samples into the PCR tubes and incubate them right before I load the gel. The instructions say to denature all the ladder in one go, but I have had better results when I denature an aliquot with my samples right before I run the chip.

Attached is a chip of 12 conifer leaf tissue extractions that worked out pretty well.

Conifer RNA Chip

 

RSS feed? (Kathryn)

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Hello people who know these things!

Is there a way for me to get an RSS feed for this blog so that I can get updates in my Google Reader? I assume you have to do something with the log in…

thanks!

Kathryn

How to post – code (Dan E.)

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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.

R script for plotting STRUCTURE results (Q values) (Rose)

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This is an R Script that plots individual Q values and labels populations. It can be modified to take average group membership from CLUMPP output and/or to import different population names and higher level groupings from elsewhere.

N.B. I haven’t run this on very many data sets, so it will probably need to be tweaked for your results. But please leave a comment if you run into any problems.

Continue reading →

Collecting tissue for DNA (Dan E.)

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

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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:
<!--more-->

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 →

Our favourite text editors (Rose)

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I hope we can start a conversation about this because a good text editor can make a big difference to a newbie, so PLEASE REPLY!!! I wanted to proselytise about Npp, but it only runs on Windows. So if you use a different OS, please make that BLEEDINGLY OBVIOUS.

Notepad++ (WINDOWS )

I’ve tried a numerous text editors over the years (like Context), but Notepad++ (Npp) is easily my favourite. It only runs on Windows, but I use it to export Unix formatted files routinely. You can set shortcut keys to change formats very easily. Npp can highlight lots of languages, including R, perl and unix. You can also define your own languages for highlighting – I did that to make my Migrate parameter files easier to read.
Continue reading →

Bioanalyzer (Kathryn)

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The bioanalyzer can be useful for quantifying and quality checking double stranded DNA, RNA, and proteins. This method of gel electrophoresis will tell you both fragment size and concentration for each fragment, using up only 1 uL of sample. The reagents and chips are expensive (for the High Sensivity chips, ~$100/11 samples/single use  chip), and have limited shelf life (4 months or less).

Rieseberg lab members have used this machine to check the quality of sequencing libraries and cDNA for microarray expression analyses. Various DNA and RNA kits are available, depending on what you want to measure, and their specs are at the bottom of this post.

Continue reading →

Moving to BC (Nolan)

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Here is a very helpful set of information on moving to BC, mainly put together by Eric Baack, and updated several years ago by Mike Barker.  One of these days we should start sending this out to people BEFORE they move to BC…

Moving to BC Barker edit

This is the most recent version (May 2014), updated thanks to Megan Stewart.

Edit (GB): looking for a place to stay? http://mapliv.com/ can have more postings than craigslist (which is also good).
 

Quantifying DNA (Dan E.)

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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 →

Filtering unmapped/unaligned reads from SAM files (Rose)

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This is a post about some time-saving help Chris Grassa gave me.

STACKS (post coming soon) doesn’t deal well with all of the unaligned reads in SAM files, so I tried using PICARD to remove them. However, PICARD doesn’t like the SAM output of BWA, but Chris G showed me how to use the Unix command awk to do it much more easily. This is his command for my file 1076.sam:
Continue reading →

If you edit a post – comment on it too! (Dan E.)

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Hello all,

Sebastien and Rose have already come across the scenario where a one RLR user has something to add to a post written by another and the sensible thing to do seems to be to edit the post.

This is fine. Editing posts is obviously the right thing to do in some situations.

The problem is that the act of editing a post is not an obvious one to anyone other than the editor. You could make a really significant contribution to a post about something very useful to the lab but this contribution might not be at all obvious to any other RLR user.
Continue reading →