Pipette Jockey

Glowing Plants 2: Fluorescent Boogaloo

Published: July 7th, 2018 Last Modified: October 16th, 2018

Ever since my original post regarding making a DIY bandpass filter, I’ve been pretty obsessed with macro-view imaging of fluorescent proteins (FPs) in plants. The quality of pictures have come a long way, take a look, it’s a leaf infected with a potyvirus that carries eGFP within its genome:

Now, maybe it’s just me, but that looks beautiful. How did I get here? Unfortunately I had to give up on using stage film, it’s cheap, you can use funny filters named “Pale Bastard Amber”, but when you’re looking at fluorescent proteins on this macro scale they don’t cut it. For a bit more money, you can get publication quality pictures with a few filters, some 3D printed parts and a cellphone camera. Let’s get into the nitty gritty logistics when it comes to looking at FPs in plants.

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Help support PipetteJockey! (Decision to monetize)

It makes me happy that people around the globe find this niche-of-niche website useful to them and their labs and I will keep this ship running as long as I can. However, the projects I do are done out of pocket on a grad student budget, so there’s a limit on the cool stuff I can do.

So, I’m deciding to monetize the website in an as non-obtrusive way as possible to get some cash flow going to fund more cool projects. Read on if you’d like to know how this will be done, in the sake of transparency between me and you as the reader.

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(PJ e7) The Dispensation Station, low cost buffer dispensing solution

Published: April 1st, 2018 Last Modified: April 2nd, 2018

Getting tired of measuring out your buffers with a graduated cylinder like an animal? Boy, do I have a project for you! Here’s a low cost buffer dispenser that you can whip up in a few days and will run you less than 100$.

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Quick and dirty bandpass filter for GFP viewing in plants

Published: January 11th, 2018 Last Modified: July 24th, 2018

Recently I had need to take macro scale pictures of plant leaves that had been infected with a virus that carries the eGFP coding sequence. Fluorescence microscopes would be the way to go, but you can only raise the lenses so far, and you’d need to remove any magnification to get a macro view. Generally, screwing around with a scope like that will make your local microscope tech pretty irate. What about a GFP illuminator that you can fit a whole plant into? These exist, but like any piece of super niche lab gear, it ain’t cheap.

How about a DIY option? Well, I’m definitely not the first to explore this area. Ian Chin-Sang and Weiwei Zhong, a couple of fellow canuks, did a write up about using LEDs and stage lighting filters to look at fluorescent proteins. Beauty, and for less than 100$ bucks! Sounds great right??? Well, once again, working with plants threw in some nice challenges!

This post will explore the implementation of their idea with a eGFP/plant/macro view use case.

(Update: Higher quality version)

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Making a Opentrons compatible liquid handling robot

Published: January 3rd, 2018 Last Modified: October 12th, 2018

It’s finally done, my very own liquid handling robot! The video goes over my trails and tribulations building this thing. Overall I’m glad I did it, the level of complexity forced me to challenge myself at every stage. This is definitely a step above assembling a ready to go 3D printer kit from ebay.

The next video will be doing some actual science with this thing…I think that was the reason to build it, right???

Thanks again to the Opentrons team for releasing their designs for us to play with 🙂

Additional improvements and pictures will go in the blog post as I make them.

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The Aliqbot, a DIY Liquid Handling Robot

Published: October 17th, 2017 Last Modified: January 31st, 2018

Pipetting hundreds of tubes worth of reagent can be hard on your hands, and on your soul. In this video I try and make my life a bit easier by throwing together a DIY liquid handling robot in the cheapest/rickety-est method possible.

This is part 1 of a multi-part series where I make this beasty do my bidding. As I mention in the video the hardest part of a liquid handling robot is not the mechanics, but the programming and control. Pt. 2 will cover expanding this concept further.

I got in touch with the people at Opentrons (https://opentrons.com/) to see if I can get their software running on my machine, which they are willing to do, yay! From what I can gather from their github page that they are running their bot from a smoothieboard of some sort, which is a very capable motion controller.

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Helios gene gun teardown and comparison to DIY model (video)

Published: September 6th, 2017 Last Modified: September 7th, 2017

For a long, long time I had lusted after the Helios gene gun. It was, as far as I was concerned, the top of the line method of bombarding your cells. Not that there was much competition, Bio-rad for the longest time was (and arguably still is) the only legit manufacturer of gene guns. At a hefty 30,000$ it’s not a piece of equipment you buy on a lark. So quite a bit of time and effort was spent in creating a DIY alternative with reasonable success.

Once in a while, though, fortune smiles upon a humble potato farmer like myself. A very generous donor actually gave me a Helios gene gun, new in box, unopened! Thank you!!! Following the teaching of our patron saint, Dave Jones, what’s the first thing I did? Turn it on? NO. TAKE IT APART!

What follows is a look at the internals of the Helios gene gun and my thoughts on it’s construction. As well, I compare it to my gene gun, which is in it’s Mk.II revision, to see just how well my gear stacks up against the pros. Lets go!

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Purifying commonly used enzymes / Homebrew pfu-sso7d

Published: August 18th, 2017 Last Modified: February 16th, 2019

Depending on the type of work you do, there may be enzymes that you go through like grad students with free cookies at seminars. For us, Q5 DNA polymerase and Superscript reverse transcriptase are two that are indispensable. Thing is, I actually don’t think these enzymes are horribly overpriced for what they are. Both enzymes are the results of years of mutagenesis and testing and are at the bleeding edge of polymerase technology. New England Biolabs, especially, charges a reasonable amount for their enzymes, Q5 included. On top of that, you can easily dilute Q5 by 1:2 to 1:4 and achieve reliable results while Superscript can be diluted to 1:8 (25U/rxn!).

Despite the reasonable prices, there are times when I think using high end, store bought enzymes are an absolute waste. You are not only paying for a fellow pipette jockey to purify the enzyme to homogeneity, but also for extensive QC and packaging. So when you take such a beauty of an enzyme and dump it into your colony PCR, well, I shed a tear no matter where you are in the world, I can sense it. I would like to see manufacturers sell two “grades” of enzymes, one that is ultra-pure for industrial/commercial users who need absolute traceability, and a lower grade for basic research use. I’m not holding my breath though, which is why we’ve spent time purifying the best equivalents to Q5 polymerase and reverse transcriptase (RT) I could get my grubby little hands on. Combined, we’ve amassed enough enzyme to last decades with a Molecular Biology Black Market (TM) value over $150,000!!! In this first post I will cover DIY pfu-sso7d and DIY reverse transcriptase will be next.

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DIY Plasmid anion exchange buffers and columns (Qiagen and Macherey Nagel)

Published: August 12th, 2017 Last Modified: December 6th, 2019

Previously we covered how to reduce cost and waste when it comes to spin kits using silica/chaotropic technologies. However, these types of kits represent the lower end of plasmid purification technologies when it comes to cost and quality of plasmid. Silica based kits, while cheaper, also have a harder time eluting plasmid DNA specifically, and you can often get genomic DNA and RNA contamination, not to mention the possibility of guanadinium salts leftover from sloppy washing.

The next evolution of plasmid kits are based on binding of the DNA to an anion exchange (AE) resin under low salt conditions, washing the resin with a mid salt solution and finally eluting with a high salt solution. These conditions allow separation of plasmid DNA from RNA and other contaminants. After precipitation the quality of DNA is superior to that of silica based kits.

Let’s take a look at the composition of the buffers for two manufacturers of AE kits, Qiagen and Machery Nagel, see how to make them, and talk about packing our own columns!

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DIY Phase Separating Gel (Phase lock) Redux: making the dense version

Published: June 21st, 2017 Last Modified: January 24th, 2019

Some time ago I wrote a post for Bitesizebio, a fellow biology themed blog, regarding the home brew version of phase lock gel. Surprisingly quite a few people found the recipe useful, and the interest has been pretty steady since then. One limitation of using vacuum grease (DOW Corning High Vacuum Grease) as phase lock is that it is equivalent to the “light” density phase lock. Light phase lock works for many day to day separations like phenol and phenol/chloroform extractions of minipreped DNA. However, for applications involving phenol + a high density salt solution, light phase lock does not work. Essentially, the aqueous phase which is supposed to be on top of your phase lock is now below it, meaning you have to poke through a layer of grease to get at your aqueous sample. The commercial manual explicitly states that light phase lock is not compatible with salty, dense solutions, and consequently they also sell a heavy version. Can we recreate the heavy version by manipulating simple vacuum grease? I think we can! Get your gloves on though, this is going to get messy.

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