“The trouble with having an open mind, of course, is that people will insist on coming along and trying to put things in it.” – Sir Terry Pratchett
Congratulations! You’ve done some, or all, of your graduate degree in Biology, which means you’re probably brimming with knowledge of peptide and nucleotide mechanics.
If you’re really lucky, you’ve also had extensive practical experience in the laboratory. If so, perhaps you should find your way across to the Experts page.
However, if you’re like me, and a shocking number of graduates out there, you’ve ended up with a shiny degree and disappointing lack of PRACTICAL EXPERIENCE.. You did the pracs, you poured the gels and then you promptly forgot the ratio of agarose to TAE. Your restriction digest went perfectly in class… but already you wonder what the name of the buffer that went with EcoRI was.
Sound familiar? Well you’re in the right place comrade. Whether you’re just starting your honours project or striking out as an independent researcher – this resource will be expressly designed to help YOU apply the knowledge of your degree where it matters most. In the lab!
If something seems super obvious and boring, skip it! This resource is intended to be comprehensive enough to fill any gaps in your knowledge, not a prescriptive step-by-step guide. YOU are the greatest expert in what YOU know, so you have my permission to skip sections at will. Now… on to the guide!
Table of Contents: Guide #3
- Pouring plates
- Mixing antibiotic media
- Mixing buffers
- Purification of Genomic DNA
- Purification of Plasmid DNA
- Agarose Electrophoresis
- Imaging and Dyes
- Sequencing prep
- Interpreting Sequencing
- Restriction Digest
- Heat Shock + Recovery
- Primer Design
- Part Design
- Functional Proteins
- Proteins for Purification
- Purification Methodology
- Logic Gates
3.1 Pouring Plates
I’m unsure how, but I finished my degree having never poured an agar dish. Most new lab members arrive with a vague memory of doing it in the first year, but from pre-prepared media. Without further ado, here is a simple protocol for LB (Luria-Bertani) media and agar.
(PROTOCOL: LB Liquid Media)
(PROTOCOL: LB Agar plate mixing and pouring)
This will be your stock standard E. coli media, but don’t be afraid to hunt down other recipes. Open Agar (Link) is likely still in development, so google “Best media (your organism)” and hunt down a recipe!
Tips & Tricks:
- Once mixed and autoclaved, you can let your LB-Agar cool and solidify. Loosen the lid and pop it into the microwave on high for 5-10 mins + ideally cool in a waterbath.
- If you don’t have an autoclave, boiling your media in a microwave will often do the trick. There are some tricky sporulated organisms that can survive this, but hopefully they aren’t living in and around your workspace
- A 60 degree waterbath is the best way to cool your media after remelting in the microwave. I especially enjoy using this method when there is a stubborn ball of agar that JUST WON’T DISSOLVE ARGH!
- Tryptone can be substituted out for peptone or milk powder in a pinch, but this may have an effect on culturability of your chosen organism.
- Likewise I’ve heard that Marmite/Vegemite can be a good substitute for yeast extract, with the aforementioned risk that it will affect culturability.
- If you’re lucky enough to have access to a laminar flow hood or biosafety cabinet, you can leave your plates exposed to dry which will result in a much less soggy plate. If not, don’t despair – the condensation inside the plate will be as sterile as the rest of your media.
3.2 Mixing antibiotic media and more plate pouring
Antibiotic media is biotech on easy mode. Just a few drops of this secret sauce is enough to purge your media of anything that doesn’t have your chosen plasmid. Well almost anything… The one caveat to this blissful state is that the mixing of antibiotic media/agar requires the one thing biologists fear most of all – maths. Worry not however! We’ve thought ahead to ensure that once you mix the tube of antibiotic stock, all maths can be left at the door.
(Protocol: Preparing Antibiotic Stock Solutions) (Mathy part)
(Protocol: Mixing Antibiotic LB-Media and LB-Agar) (Haha, all the maths was done earlier part)
3.3 Sterile Technique
Okay you’ve just made some really tasty microorganism food, now you must learn to defend it from the wider world. Sterile Technique is more than just jargon, it’s a way of life. It’s also the first thing a nosy onlooker will comment on – knowing WHY is just as important as learning HOW. It can feel superfluous when working with antibiotics and bacteria (because easy mode) but I recommend you go through the motions anyway. Though failure at sterile technique can often yield fascinating and bewildering results; I’m sure that Alexander Flemming and Bob Ross would agree that “we don’t make mistakes… just happy little accidents”
3.3.1 Choosing your organism – SKIP if you’ve already chosen a target
Want to know something extremely unsexy about synthetic biology? For most of the time, the sum total of your life’s work will look to the layman like this:
If you imagined that this guide was going to let you give yourself super strength, or create a glowing rabbit – TURN BACK NOW. Soon, maybe, later… we will have the power to manipulate genetics with a flick of the wrist, but we aren’t there yet. Legal and ethical reasons aside, our understanding of the sheer complexity of life is still nascent. You’re going to want a simple, single-celled organism to work with.
I personally recommend anything that can hold and utilise a plasmid, such as Bacteria or Yeast. It is important to choose a non-pathogenic strain and try to source a well characterised strain from a respected laboratory or supplier.
Choosing a single-celled target allows you far more power of manipulation over DNA at this stage of technological development. This guide will not involve many In Vivo experiments, instead we’ll be slaughtering our bacteria in order to extract the sweet, sweet plasmid DNA within. The power of In Vitro synthetic biology is that we can separate DNA and RNA for manipulation without worrying about killing the long-deceased bacteria.
Now read the above paragraph, but replace the word “bacteria” with the word “bunny rabbit”, and this should help you understand why you won’t be working with higher-order mammals. If not, you’re a monster and I respect your honesty.
A very important step while choosing your target organism is legality. I can’t answer this for every country, so spend some time googling your government’s stance on genetic manipulation. The language of these laws can be quite dated – such as Australia’s Gene Technology Act (2001): so if you’re worried, try calling your country’s regulator, such as Australia’s OGTR. The well trod path will often be the easiest answer (K12 derived E. coli strains) but worry not; we will teach you to do miraculous things in this admittedly dull-to-the-naked-eye organism.