Biotech bench – transformation, restriction digest and gel
BiologyBiotechnologyAges 17–18
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Sign in to playTwo gene-technology practicals on one virtual bench. Transform E. coli with a plasmid carrying an ampicillin-resistance gene and an arabinose-inducible GFP gene (CaCl₂, ice, 42 °C heat shock, recovery), spread LB, LB/amp and LB/amp/ara plates, incubate in time-lapse, count colonies, check them under UV light and calculate the transformation efficiency step by step. Then digest phage DNA and the plasmid with EcoRI, BamHI and HindIII, load and run an agarose gel, stain it, measure band distances, build a log(bp) standard curve from the DNA ladder and read off fragment sizes.
Lesson: Genetic engineering: bacterial transformation, restriction enzymes and agarose gel electrophoresis of DNA
What it shows
Genetic engineers move genes into bacteria and check DNA by cutting it. In the transformation tab, CaCl₂ and a 42 °C heat shock let E. coli take up a plasmid with an ampicillin-resistance gene (bla) and a GFP gene switched on by arabinose. Only transformed cells grow on ampicillin, and they glow green under UV only when arabinose is present. In the gel tab, restriction enzymes cut DNA at their recognition sequences; the negatively charged fragments move through agarose towards the positive electrode, and smaller fragments travel farther. A standard curve of log(size) against distance for a DNA ladder gives the sizes of unknown fragments.
How to use
In 1. Transformation, press the step buttons in order, or use Skip this step to leave out ice, heat shock or recovery. After Incubate at 37 °C, set the incubation time, switch on UV light, tap a plate to enlarge it and tap colonies to count them; the efficiency calculation fills in. In 2. Digest and gel, choose DNA and enzymes for each lane, then Digest at 37 °C, Load wells, Run gel and Stain and view. Tap bands to measure them and read sizes from the standard curve.
Parameters you can change
- Opening tab Bacterial transformation, Restriction digest and gel
- Plasmid concentration 0.02–0.2 µg/µL
- Heat-shock time at 42 °C 0–120 s
- Volume spread on each plate 50–250 µL
- DNA to digest Linear phage DNA (20,000 bp), Circular GFP plasmid (5,400 bp)
- Gel voltage 50–150 V
- Gel run time 10–90 min
Questions to explore
- Why do cells without the plasmid form a lawn on LB but no colonies on LB/amp?
- Why do colonies glow under UV on LB/amp/ara but not on LB/amp, although both plates have similar counts?
- How many fragments do three cuts give in linear DNA, and how many in a circular plasmid?
