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!

Passing DNA through a silica column twice does not increase yield – A cautionary tale of cognitive bias

Scientists are stereotyped as objective thinkers, putting hard facts above our own subjective experience. If you’re being honest though, as a group of people we’re particularly vulnerable to a host of cognitive biases including magical thinking, confirmation bias, etc. Why the maudlin tone??? Well, I challenged my own magical thinking, and it turns out I was wrong. Not just a little wrong, but pretty definitively wrong. Oh well, better to don the dunce cap for one day, than be a dunce forever.

Lab Primer Class 1 Lecture 1 – Making a useful protein A to Z

Published: December 30th, 2019 Last Modified: January 17th, 2020

Just trying my hand at educational content, no holds barred, no details spared.

In this lecture our goal is to purify a useful protein from start to finish! I cover reading patents, verifying sequences, designing the experiment, designing primers, doing the PCR, cloning and screening. Part two will be the induction, expression and activity testing of the protein.

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!

TEDA Cloning – Cheap, easy Gibson alternative

Published: November 22nd, 2019 Last Modified: November 25th, 2019

Manipulating plasmid DNA (which we’ll colloquially call cloning) is one of the hallmarks of modern molecular biology. Yeah yeah, I hear you, gene synthesis is coming to take our jobs away, it will render our cloning skills useless, etc etc. Keep your shirt on! Even if DNA synthesis rates keep dropping often it’s more cost effective AND faster to do some or all of the assembly of the fragments yourself.

Now, we’ve come a long way from when your grandmammy or grandpappy synthesized their own primers and purified restriction enzymes to make the first plasmids. My first forays into modern cloning techniques hopped from ligation independent cloning (LIC) to sequence and ligation independent cloning (SLIC) and finally settling in to Gibson assembly as my method of choice. You have a mastermix, you mix it with the DNA you want to assemble, you transform it, et voila! You (hopefully) have your plasmid.

Problem is, commercial 2X Gibson mix is often between 10-20$ per reaction, and the homemade stuff can only be made at about 3$ per reaction. On top of that, Gibson mix has a notoriously poor shelf life. I have good news folks, you can now make a drop in replacement for Gibson mix at less than a CENT per reaction!

More plasmids, hooray!

Published: November 12th, 2019 Last Modified: November 22nd, 2019

Already purified pfu-sso7d? Got a lifetimes supply of Mashup-RT in the freezer? Still hungry for more??? Don’t worry, I have you all covered. With gene synthesis rates plummeting we can move past purifying the low hanging fruit and get into some niche, but useful, enzymes.

Check out the Plasmid List to see what’s on offer!

I am introducing two enzymes to the lineup: DpnI and Murine RNase inhibitor (MRI).

If you spend more than a couple hundred dollars a month on either enzyme it is certainly worth the effort to purify your own. DpnI is extremely useful in cloning/PCR heavy labs for digesting plasmid DNA after a PCR reaction is complete. MRI is used in reverse transcription and any reaction where you suspect possible RNase contamination. Purifying your own can not only save you money, but you can find new applications for the enzymes where it was cost prohibitive before.

Purification protocols are coming, beta testers please apply!