Basic components of genotyping:
- The engineered primer (the hard part): a genetic sequence responsible for a measurable/observable phenotypic trait (we're going to presume you have been provided 5' to 3' sequence that must be fabricated into a primer sequence that needs to be ordered.
- A restriction enzyme (if applicable, more on the purpose of restriction digest later on): variable temperature water bath
- Equipment:
- 0.5-2 µL or 0.5-10 µL, 20-200/300 µL, 100-1000 µL micropipette men for measuring out liquids
- 1.5/1.7 mL sterile (DNA/RNA/DNAse/RNAse-free tubes)
- 0.2 mL PCR tubes with caps. I recommend the strip of tubes that are connected together
- A PCR tube cassette (for holding your tubes when preparing/loading/keeping them organized.
- A thermal cycler for amplification
- A shaker/rocker
- A gel box w/ combs
- A multi-variable power-supply with leads (I don't repurposing one not designed for this use)
- A vortexer (for mixing up your samples)
- A dry incubator
- A 1.5 mL tube rack
- A dedicated microwave (not used for food)
- Two food-grade containers for ethidium bromide developing baths
- An electric stirrer/heating plate
- A computer, imaging box w/ camera and UV light, and Sony CareStream printer or equivalent
- A box of nitrile gloves
- A bucket with lid (for disposing of toxic gels)
- Supplies:
- A tail lysis buffer/sample lysis buffer (in the case of higher biosafety level and human communicable agents, for instance COVID, it is recommended to heat-inactivate the same before handling/use)
- Promega GoTaq Green, DreamTaq, or any other 2x master mix of your choosing
- NFH2O (nuclease-free H2O)
- TAE: Trisbase, glacial acetic acid, EDTA (or purchase pre-made 50x, or 1x; if performing dilutions of 50X, you will need minimally deionized/purified water - I don't recommend tap water)
- Ultra-pure agarose (or make your own)
- ethidium bromide (powder, or already target/mixed stock concentration of 10mg/mL)
- Clean 1 L bottle w/ magnetic stirrer bar (for preparing 50xTAE)
- Another 2L clean bottle
- A sample of DNA: usually in the form of mouse: tail, or ear-punch in an animal research setting; or saliva, tissue/blood sample from a subject/patient in a clinical setting.
WARNING: ethidium bromide as a reagent intercalates DNA, meaning it binds to DNA (including your own!) and is therefore a known mutagen! For this reason, exercise extreme caution when working with it, or better yet use something less toxic, like SYBR green, instead. Unfortunately, since I have never worked with the SYBR reagent, I cannot provide directions on its use as a substitute for ethidium bromide to get your gels to illuminates.
Let's begin with the concepts first, as it helps immensely to understand why qualitative PCR along with gel electrophoresis is still useful to this day, even if quantitative PCR is taking over most of the market.
Concept
Polymerase chain reaction (PCR) involves creating a reaction mixture that consists of the following: a forward and reverse primer, a heat-activated taq enzyme (which copies the sequence), glycerol (later allows samples to sink in water and into the wells), base pairs, MgCl, and nuclease-free H2O (to lower the viscosity and enhance...
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