Author: Pipette Jockey

Mashup RT Update #2 – Our Take on Purification/Activity Assays/Buffer Optimization

Published: July 15th, 2019   Last Modified: April 16th, 2020

It’s awesome how far we’ve come as a little community in less than a year, from the first inception of Mashup to the first feedback from the beta testers. It’s finally time that I present our take on it’s purification as well as characterization of it’s activity and improvements to the RT buffer.

Considering that Mashup is now fully integrated into our lab’s workflow, I can comfortably call this project a success. Grab a coffee, get settled in, let’s get to all the juicy details!

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PSA: Your thermal cycler is NOT a refrigerator (Don’t hold below ambient!)

Published: June 10th, 2019   Last Modified: August 27th, 2019

One of the most precious machines in the molecular biology lab is the thermal cycler, often called the PCR machine. They dutifully cycle between the temperatures you set, amplify your DNA and never complain. We’ve got quite a herd ourselves and we love them all dearly. We’ve got Rowan, Cycler #2, Chad, River, Ol’ Red and Greybeard.

Unfortunately, these machines are often not treated with the respect of a 20,000$+ piece of gear and are instead used as a glorified mini-fridge. What do I mean by that? After your PCR is finished, do YOU set it to hold at 4C? Do you let it hold 4C overnight? Do you let it hold at 4C on a Friday and only turn off the machine Monday? Do you compromise and let it hold at 10C or 16C? Stop! Just stop!

There’s NO advantage to holding your PCR reaction at anything below ambient for any length of time! Let me try to convince you so you can SIGNIFICANTLY extend the lifetime of your machine.

This message is brought to you by the People for the Ethical Treatment of Thermal Cyclers (PETTC)

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Mashup RT Beta Update #1

Published: April 19th, 2019   Last Modified: February 19th, 2020

Mashup Reverse Transcriptase (RT) was released less than half a year ago and many researchers have come forward to become beta testers. I am eternally grateful to everyone who took their time, effort and funding to express Mashup-RT.

I am pleased to say that due to the efforts of you wonderful people that the project is quickly maturing. Mashup-RT is not only active, but seems capable of holding its own against commercial offerings, which was the original objective.

This post is a compilation of feedback/purification protocols I’ve received, so that new testers can have a better starting point than I have in my original post, which is outdated. As well, I will be posting results of my own purification, which is coming soon.

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(PJe10) Make re-usable spread sticks // inoculating loops // hole cutters

Published: April 15th, 2019   Last Modified: April 21st, 2019

Hands itching, got some free time, want to make some something useful? Here’s a three part video on how to make re-usable bacterial spread sticks, inoculating loops and tissue hole cutters! As well, I give you a easy, cheap system to sterilize and re-use them!

(PJe9) Make your own media premix // How to microwave agar plates

Published: March 29th, 2019   Last Modified: April 21st, 2019

Make a lot of media? Don’t want to drag out your 2 kilo jars of tryptone, yeast extract and salt every time? Make some pre-mix quickly, cheaply and easily!

Also, learn the dark art of microwaving agar plates. Just a trick to keep in the back of your pocket for when things don’t go according to plan.

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Dense Phase Separating Gel / Homemade TRIzol combo

Published: November 13th, 2018   Last Modified: April 21st, 2019

Homemade phase lock gel, which we can generically call phase separating gel (PSG), can be a useful addition to your DNA extraction repertoire. The best part is that for many applications you can use 100% DOW High Vacuum grease, which makes it very economical. Unfortunately, for RNA extractions with TRIzol, the stock PSG is not dense enough to migrate between aqueous and organic phases, and simply settles on top of your aqueous phase. BOO!!! 

Now, I’ve been trying to solve this problem for a while, but this is the first time I think it’s been successful. Basically, by combining vacuum grease with 15% w/w silicone dioxide (i.e. 20g grease + 3g SiO2), you end up with PSG dense enough to work properly with TRIzol. Lets take a look at how to do it with a minimum of mess!

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A quick teardown of the Beckman Biomek P1000 Pipette Module

Published: November 2nd, 2018   Last Modified: November 14th, 2018

Some time ago I was pretty obsessed with building a pipetting robot, and to a degree it was a success. However, building it burnt me out pretty good. I will revisit the project and likely rebuild the whole thing to make it simpler and closer to the original plan. I tore down my opentrons clone, and this is all that remains:

Ahh, memories. But let’s not get all nostalgic and wax poetic. One aspect of the original opentrons robot (which has now been corrected, see their OT2) was that the pipetting section used a hand operated pipette strapped to some stepper motors. I was looking for inspiration from the pros to make my own pipetting module, so I got my grubby little paws on a Biomek P1000 module to tear down. Take a look, it’s a rare treat to see how a part of a 100,000$+ machine is made!

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MashUp-RT, purify your own reverse transcriptase (Beta testing phase)

Published: September 6th, 2018   Last Modified: April 21st, 2020

Purification of pfu-sso7d ended up being a stunningly popular article, only being beaten out by DIY decontamination mix (people really enjoy decon mixes, apparently). What it revealed was that there is a global demand by scientists who wish to purify their own enzymes to be used as tools in their labs. I’m glad that I’m not the only one who has adopted the DIY ethos.

Another detail that surfaced is that besides an enzyme useful for PCR, a reverse transcriptase (RT) was in equally high demand. This isn’t surprising when you consider that RTs are some of the most expensive enzymes for RNA biologists and if you’re between funding cycles, lost your funding or not in a central country it can be a barrier to entry/continuation in the field. So, you asked, and I have delivered: MashUp (MU)-RT, a custom, FeLV based RT available to academic users!

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