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

Published: August 14th, 2020   Last Modified: August 19th, 2020

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!

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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:

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

Let me show you how!

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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!

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

Published: April 9th, 2020   Last Modified: April 14th, 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!

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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).

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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!

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!

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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.

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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.