4 ms·

Some issues and general observations: * Making plates is hard. You really need an autoclave, and a laminar flow hood is also very helpful. For a DIY approach

Some issues and general observations: * Making plates is hard. You really need an autoclave, and a laminar flow hood is also very helpful. For a DIY approach, you need a pressure cooker and a bunsen burner (in a relatively still room, the bunsen burner creates a radius of sterile space around it. Yes, this is legitimate lab technique, if a bit old-school). Those plates were clearly contaminated, and without proper sterile technique, nothing whatsoever is going to work. The kit's instructions, in my view, are unlikely to produce sterile plates. They provide sterilized agar, but need to be poured in a sterile area, but make no provision for this. * The instructions for the kit are just barely sufficient. There are a million different ways you can do things incorrectly, so very clear and thorough steps are needed for a novice to have a chance at success. A lot of troubleshooting material should be included as well. This is a setup for failure in my view. For instance, an un-inoculated control plate is a bare minimum control. For a HN-friendly analogy, it would be like debugging software without any error reporting, print statements, etc. * Lab equipment is mostly fridges, water baths, plastic disposables, and moderately specialized stuff like tabletop centrifuges and thermocyclers. It seems relatively accessible, which gets people excited about DIY microbiology, but it's deceptive. Temperatures, and the stability of temperatures, can matter a lot. Reliable equipment is very important. Someone seriously looking to do this (it's certainly possible!) needs to be able to devote a fair amount of space and resources to their home lab. * The #1 difference between a 'real' lab and a home lab is access to troubleshooting resources. Shit goes wrong all the time, and experienced post-docs, lab techs, colleagues, etc. are what make research possible. You also very often need access to quality materials for positive and negative controls. It's not uncommon for entire labs to grind to a halt for weeks or months until some tricky bit of troubleshooting is resolved. Some people are commenting that this isn't 'CRISPR'. It is. The transformation provides a plasmid that will not convey resistance on it's own (unless they're being dishonest about what's in the kit). Only when a stable transformation of the genome occurs will resistance be seen.

dekhn 8y ago
I spent a year in a lab learning how to clone genes into E.Coli. it's amazing how easy it is to screw up. Once, we got a bottle of broth back from the autoclave (it should be completely sterile) and within a week or so of sitting on the shelf, unopened, there was already a cloudy membrane growing inside. What I learned from the process is that it's all about control experiments and troubleshooting. To make a real discovery today, or even just convince yourself you succeeded in a process, you need to be outright paranoid about every component in your system. I wasted a lot of time because I started with only negative controls, and learned the hard way that you need positive controls. That doesn't even begin to describe all the problems I had extracting reliable data from gels. I'm convinced that all the dark secrets in biology are in the gels. Even when I worked for a major biotech company, when i talked to the guys who ran the continuous fermentors said they always saw strange fluctuations in things like bulk metabolic rate, drug production, etc, that they couldn't explain, and they were the experts in reliably growing liters of e.coli.

dekhn 8y ago

I spent a year in a lab learning how to clone genes into E.Coli. it's amazing how easy it is to screw up. Once, we got a bottle of broth back from the autoclave (it should be completely sterile) and within a week or so of sitting on the shelf, unopened, there was already a cloudy membrane growing inside. What I learned from the process is that it's all about control experiments and troubleshooting. To make a real discovery today, or even just convince yourself you succeeded in a process, you need to be outright paranoid about every component in your system. I wasted a lot of time because I started with only negative controls, and learned the hard way that you need positive controls. That doesn't even begin to describe all the problems I had extracting reliable data from gels. I'm convinced that all the dark secrets in biology are in the gels. Even when I worked for a major biotech company, when i talked to the guys who ran the continuous fermentors said they always saw strange fluctuations in things like bulk metabolic rate, drug production, etc, that they couldn't explain, and they were the experts in reliably growing liters of e.coli.