Category: Reverse Engineering Reagents/Recipes

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!

Published: July 15th, 2019   Last Modified: April 16th, 2020

It’s awesome how far we’ve come as a little community in less than a year, from the first inception of Mashup to the first feedback from the beta testers. It’s finally time that I present our take on it’s purification as well as characterization of it’s activity and improvements to the RT buffer.

Considering that Mashup is now fully integrated into our lab’s workflow, I can comfortably call this project a success. Grab a coffee, get settled in, let’s get to all the juicy details!

Published: August 18th, 2017   Last Modified: February 16th, 2019

Depending on the type of work you do, there may be enzymes that you go through like grad students with free cookies at seminars. For us, Q5 DNA polymerase and Superscript reverse transcriptase are two that are indispensable. Thing is, I actually don’t think these enzymes are horribly overpriced for what they are. Both enzymes are the results of years of mutagenesis and testing and are at the bleeding edge of polymerase technology. New England Biolabs, especially, charges a reasonable amount for their enzymes, Q5 included. On top of that, you can easily dilute Q5 by 1:2 to 1:4 and achieve reliable results while Superscript can be diluted to 1:8 (25U/rxn!).

Despite the reasonable prices, there are times when I think using high end, store bought enzymes are an absolute waste. You are not only paying for a fellow pipette jockey to purify the enzyme to homogeneity, but also for extensive QC and packaging. So when you take such a beauty of an enzyme and dump it into your colony PCR, well, I shed a tear no matter where you are in the world, I can sense it. I would like to see manufacturers sell two “grades” of enzymes, one that is ultra-pure for industrial/commercial users who need absolute traceability, and a lower grade for basic research use. I’m not holding my breath though, which is why we’ve spent time purifying the best equivalents to Q5 polymerase and reverse transcriptase (RT) I could get my grubby little hands on. Combined, we’ve amassed enough enzyme to last decades with a Molecular Biology Black Market (TM) value over $150,000!!! In this first post I will cover DIY pfu-sso7d and DIY reverse transcriptase will be next.

Continue reading “Purifying commonly used enzymes / Homebrew pfu-sso7d”

Published: August 12th, 2017   Last Modified: December 6th, 2019

Previously we covered how to reduce cost and waste when it comes to spin kits using silica/chaotropic technologies. However, these types of kits represent the lower end of plasmid purification technologies when it comes to cost and quality of plasmid. Silica based kits, while cheaper, also have a harder time eluting plasmid DNA specifically, and you can often get genomic DNA and RNA contamination, not to mention the possibility of guanadinium salts leftover from sloppy washing.

The next evolution of plasmid kits are based on binding of the DNA to an anion exchange (AE) resin under low salt conditions, washing the resin with a mid salt solution and finally eluting with a high salt solution. These conditions allow separation of plasmid DNA from RNA and other contaminants. After precipitation the quality of DNA is superior to that of silica based kits.

Let’s take a look at the composition of the buffers for two manufacturers of AE kits, Qiagen and Machery Nagel, see how to make them, and talk about packing our own columns!

Continue reading “DIY Plasmid anion exchange buffers and columns (Qiagen and Macherey Nagel)”

Published: June 21st, 2017   Last Modified: January 24th, 2019

Some time ago I wrote a post for Bitesizebio, a fellow biology themed blog, regarding the home brew version of phase lock gel. Surprisingly quite a few people found the recipe useful, and the interest has been pretty steady since then. One limitation of using vacuum grease (DOW Corning High Vacuum Grease) as phase lock is that it is equivalent to the “light” density phase lock. Light phase lock works for many day to day separations like phenol and phenol/chloroform extractions of minipreped DNA. However, for applications involving phenol + a high density salt solution, light phase lock does not work. Essentially, the aqueous phase which is supposed to be on top of your phase lock is now below it, meaning you have to poke through a layer of grease to get at your aqueous sample. The commercial manual explicitly states that light phase lock is not compatible with salty, dense solutions, and consequently they also sell a heavy version. Can we recreate the heavy version by manipulating simple vacuum grease? I think we can! Get your gloves on though, this is going to get messy.

Continue reading “DIY Phase Separating Gel (Phase lock) Redux: making the dense version”

Published: June 16th, 2017   Last Modified: November 23rd, 2019

Silica based nucleic acid purification columns and kits are a common consumable in the lab. They’re used for cleaning up PCR reactions, purifying gel slices and plasmid DNA minipreps. Inevitably, when you reach the bottom of the box you are usually left with one of two scenarios: 1) You don’t have enough of a specific buffer or 2) You don’t have enough columns. Both of these scenarios usually result in the remaining components of the kit being tossed out or forgotten in your cupboard for a decade (that somehow make their way into my hands).

Why not source your own columns, mix your own buffers to both reduce waste and save money! Grab a snack, ease into your bunny slippers, this is a long one.

Continue reading “Don’t throw leftover silica DNA purification columns/buffers away! Use them up! (DIY Silica Spin Kits)”