Pipette Jockey

Complete, free Genomics course (Lecture notes, recordings, assignments and tests!)

Published: July 31st, 2022 Last Modified: November 12th, 2022

During the fall of 2020 I had the opportunity to design and teach a Genomics course for 60+ 4th year undergraduate biology students. It was one of the harder (but also most rewarding) things I’ve had to do, especially with 3 months to prepare. I’m proud of the course I delivered and I’d like to share ALL my class materials so that the next person has an easier time than I did 🙂 As well, the field of Genomics moves so fast I don’t want it to be completely out of date before somebody gets some use out of it.

Click read more for links to the rationale behind the design of the course, caveats, credits, etc!

Introducing Synbio Studio at cell-free.com!

Over the last year I’ve been having an absolute blast developing reagents and hardware for the synthetic biology space. I am proud to announce the launch our company store / blog at cell-free.com. Our primary focus is on advancing the field of cell-free protein synthesis through higher quality, more accessible reagents coupled with hardware that helps you get the most value out of your cell-free and molecular biology experiments.

Why are we so excited about cell-free protein synthesis? Gone are the days of rabbit retic lysate or wheat germ extract, with yields of 20 ug/mL or less. Modern E.coli lysate based systems are capable of producing more than 1.5 mg/mL of protein! Drop your template DNA into the reaction, mix a few reagents and collect your protein in 4-16 hours. When compared to the cost of reagents and labor to produce a similar amount of protein in E. coli, cell-free protein synthesis has the potential to replace small scale cell-based protein synthesis in many labs.

Along the road to launch we have developed tools that we will be sharing as open-source projects on our blog. One that I’m particularly excited about is our in-house bag bioreactor which helps us test feeding strategies for our cells before scaling up! I’ll be sure to let everyone know as soon as it’s up. Thank you for all the continued support everyone.

Life Changes – Halting Plasmid Distribution

As some of you may know I recently obtained my PhD 🙂 and have been looking for work. I’m very happy to say that a wonderful opportunity has come up! I will be starting work at a biotech company on the microfluidics / molecular biology side!

As I transition to this new non-academic world, I’m voluntarily deciding to halt distribution of the plasmid pack. I will fulfill requests made up to today (Feb 16th, 2021), however going forward I will have to decline further requests. This is to avoid any potential conflicts of interest and to focus myself for the tasks at hand.

The blog will continue to grow and mature, this is a labor of love and I appreciate meeting all the wonderful DIYers and scientists around the globe.

Cloning Workshop

Published: January 5th, 2021 Last Modified: January 7th, 2021

I’ve been slowly chipping away at a series of educational videos focused around DNA manipulation and cloning. I’ve taken into account the feedback from the last educational video, so things are a bit more streamlined this time!

I will be posting powerpoint slides, protocols and resources on this page as an accompaniment to the videos! Hope you enjoy! 🙂

New Plasmids!!! Cheeky TEV, MBP-Sumo/TEV-MRI, HIS-Taq

Published: August 14th, 2020 Last Modified: September 30th, 2021

Apologies for the hiatus and slow replies everyone, I’m entering one of the busiest parts of my life (to date…) and I will hopefully have a nice surprise update in a few months 😉 During these busy times I often turn to the lab bench as a sort of stress ball, and I figured it was time to share a few things I’ve been fidgeting with.

This release is probably the biggest to date with 4 new plasmids! My hope is that these enzymes will fill some holes in the current lineup. The ultimate goal is a small, curated pack of plasmids that are broadly applicable to molecular biologists.

1) Cheeky TEV (CTEV) – Everyone and their cat has the old-school TEV plasmids at this point, but there’s a whole world of TEV variants to explore and here’s my cheeky version of the current state of the art!

2)HIS-MBP-TEV-MRI and HIS-MBP-Sumo-MRI – My pML-MRI plasmid is being replaced by two superior versions generated by long time contributor Ye Yang. He took the ORF of my MRI and sub-cloned it into his vectors. Thank you! These variants will be easier to purify and give a higher soluble yield.

3) His-Taq – For specific assays Taq polymerase is preferable to something like pfu-sso7d. Generally it’s pretty difficult to find a HIS-tagged Taq polymerase, so here we are, nice and simple!

Read on for rationale and further details. Academic users can get in contact to test the plasmids!

Sending plasmids the easy way, paperless!

Some time ago I did a post about the stability of plasmids stored with and without filter paper. Basically, plasmid DNA stored on filter paper has a shorter shelf life than just plasmid in a lil’ plastic bag. As well, the addition of trehalose further increases dried plasmid stability. Thing is, I never actually described HOW to make lil’ plastic bags filled with plasmid. Whoops! Can’t really expect people to adopt a method if there’s no protocol, eh?

Why would you switch over to paperless? If it was good enough for grandma/grandpa, isn’t it good enough for me?? Paperless is:

Faster and scalable, if you’re sending one letter or fifty!
Less handling, therefore less chance of cross contamination!
Longer shelf life!
Much easier for the user to recover the DNA!!!

Let me show you how!

Making (and using) a vacuum manifold!

Published: June 20th, 2020 Last Modified: June 23rd, 2020

We all know the sheer joy of doing just a handful of minipreps at a time. You pull out all the flicks of the wrist and bits of finesse you’ve built over the years. Once you move past…oh…8 minipreps, and it becomes work. After 16 or 24 minipreps and you’re into re-evaluating your life choices. Those wash steps add up! What about midi-preps? Maxi-preps? Protein columns? Yeah, being able to process those in parallel in a efficient manner sure would be nice. In comes….THE VACUUM MANIFOLD!!!!

What does the beastie do? You apply a vacuum at the barbed connector, open the valve, and the vacuum is distributed between the little inlets, allowing you to suck away 16 columns worth of wash buffer at once! It’s not the cheapest build at 55$ worth of parts and access to a 3D printer, but considering alternatives start at 150$ and go into the hundreds, it’s worthwhile (and fun!) to build your own. Let’s go!

Store your bacteria and plasmids dry at RT with the magic of trehalose! No -80 needed!

Published: April 9th, 2020 Last Modified: November 5th, 2020

For many people, their -80C freezer is one of the most critical pieces of lab equipment. It can be your life’s work, all packed up in 3x3ft footprint. Your glycerol stocks, competent cells, tissue samples etc. Getting a call that your -80 is in a rapidly expanding puddle of water is no fun, to put it lightly.

We put up with it, however, because what other choice to we have? As it turns out, quite a bit! There are a few ways in which you can lessen your dependence on the -80, and one that I’ve been investigating is preserving E.coli and plasmid DNA dry at room temperature! Let’s see how you can add this trick to your sample preservation toolkit!

DIY RNA Spin Column Buffers – Purification of RNA with humble DNA silica spin columns

Published: March 30th, 2020 Last Modified: April 20th, 2020

Personally I don’t have much experience with RNA spin columns. We’re more of a Trizol/RNAzol lab ourselves (DIY recipe for Trizol, thanks Julian, wherever you are). However the convenience and speed of spin columns cannot be denied. Unlike DNA silica purification, there is less known about brewing your own buffers.

I’ve put off reverse engineering these recipes, but I think it’s finally time. This page is a work in progress, I’ll update it when I have data over the next little while. Readers are encouraged to try the buffers themselves and leave a comment, or get in touch so I can put up your data (With full credit, of course).

Mashup RT is available for academics and health care providers

Published: March 21st, 2020 Last Modified: April 16th, 2020

I’ve got the Mashup RT plasmid (and everything else on the list) to send out to academics/health care providers running out of commercial enzymes and kits, get in touch through the contact form.

!!! Most up to date Mashup-RT Purification Protocol !!!
PDF, DOCX

V1.2 Update:
– DTT should be added to final concentrated protein at a concentration of 1mM. My mistake, please accept my apologies. Dropped by accident, thank you kind reader for informing me.

V1.1 Update:
– Added section for FPLC users
– Added representative gels of purification
– Bumped up imidizole in Lysis Buffer from 20 to 40mM
– Bumped up imidizole in Wash buffer from 50 to 80mM

FAQ:

Yes, Mashup is thermostable, active up to 70C, albeit at a reduced activity. Typical reaction temperatures are 50-55C
The yield from 1 liter of culture is 250000 to 500000 reactions
My concentrated stock is approximately 0.5 mg/mL, going higher than this will trigger precipitation
Quantify Mashup with the BCA assay, Bradford does NOT work

Please leave corrections/additions in comments, thank you!