Reverse Engineering Reagents/Recipes

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!

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!

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! ***

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!