Uncategorized – Page 2 – Pipette Jockey

p50-T4 DNA Ligase Plasmid – Brought to you by Chemically Incompetent!

Published: February 5th, 2020 Last Modified: April 14th, 2020

One of the perks of running this site is meeting all sorts of interesting characters around the globe. Now and again you meet someone who really aligns with your values and one such fellow is Mark, who runs Chemically Incompetent. I highly encourage everyone to check out his website, he does an excellent job of blending science, philosophy and humor in his writing. If you like saucy lab hacks and ways to improve your day to day efficiency, then it’s right up your alley.

Mark has been working on a plasmid which encodes an improved version of T4 DNA Ligase fused to the p50 domain. This fusion is an upgrade over the venerable T4 Ligase and should save you heaps if you do massive amounts of cloning or you make your own Gibson mix.

He’s done a lot of work on the writeup and purification protocol, it’s clean and thorough. I’m happy to announce that he has allowed me to carry the plasmid as part of my plasmid pack. I will add my take on the purification protocol as soon as I give it a whirl! Thanks Mark! 🙂

Hysterisis Hysteria – How good is your incubator for reals??

Published: January 31st, 2020 Last Modified: April 14th, 2020

Do you have a favorite heat block in your lab? One that you avoid like the plague? Are you a religious believer in the nirvana-like stability of the water bath? Well folks, have I got some food for thought for you! Simply sticking a thermometer in your block/bath doesn’t tell the whole story when we talk about stability or accuracy of an incubator. There’s the almost invisible dimension of time and little minutia like probe setup and placement. What the hell am I talking about? You know, it’s easier to explain with a graphic, here’s what our old analog heat block looks like when you graph the temperature over about 6 hours:

Sweet merciful baby Jesus, that’s a lot to take in. We’re getting overshoot, all sorts of temperature fluctuations, other madness! Let’s get into it!

Long term plasmid stability on paper + new paperless method

Getting new plasmids in the mail is like Christmas, there’s nothing like the anticipation of cracking open a letter and adding a new enzyme or technique to your toolbox. The logistics of non-profit plasmid distribution can be tedious at times, but getting feedback about how your labs made buckets of enzymes and saved thousands of dollars make my day 🙂

Now, I ain’t Addgene level of fancy, but I do try to keep things classy. I always thought that the classic “plasmid on filter paper” method was adequate for sharing plasmids. Just like nonna used to make! I even vacuum pack them to reduce the chance for cross contamination. The question always stuck with me though, how long does DNA last on paper, and is there a better way to do things?

I finally cracked the seal on a 1.5 year time trail of plasmid DNA on paper and the results are fascinating! Not only that, but I think we have a far superior way of sending plasmids now!

Re-using Electroporation Cuvettes (Without autoclaving!)

Published: December 13th, 2019 Last Modified: January 29th, 2020

Love that high transformation efficiency of electroporation, but hate throwing away cuvettes like they’re going out of style? No more! Wash those suckers out and save some serious money.

Transformation a Go-Go (TGG) Protocol + Twitter

Published: November 29th, 2019 Last Modified: December 13th, 2019

Getting the social media (“sosh meed”) train rolling to help grow the DIY Biology community. Follow me to get regular updates on when I post something new! And what better way to kick things off than to share a sneaky little protocol with you all?

Let’s say you’re doing a standard transformation to get a plasmid into BL21s for protein expression, or to get fresh colonies for a maxi prep. You’ve just had nice, fat burrito and the hour and a half protocol could be better spent on a nap. Skip all that! Heat shock your cells and plate just a whiff, you’ll get enough on your plate to do what you need to do. You only need one colony after all.

Competent cell protocol from Wendy Chao’s blog, thanks Wendy, wherever you are 🙂

*** Do not use this protocol for cloning reactions where you expect low efficiency, like a 4 fragment Gibson assembly/ InFusion or the like. Those experiments deserve the extra hour and a half finesse steps, you need every edge you can get! ***

Cheat Sheets / Calculators

Published: November 23rd, 2019 Last Modified: December 13th, 2019

Cognitive load already high? Don’t feel like doing calculations or digging out your protocol binder from under the test tube racks and despair? Lighten your load with some easy to reference protocol cheat sheets and spreadsheets that do the work for you!

Plasmid Prep cheat sheet, Silica column cheat sheet, PCR master mix calculator and MORE!

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)

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.

(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.