In Recombinant DNA a gene was cut and pasted into a plasmid. The next step is getting the plasmid into bacteria and finding the ones that took it up. Then, to be sure what you have, you read the DNA: sequencing. Sizing the pieces on a gel is part of both.
- Transformation: bacteria take up DNA from their surroundings. In the lab, a calcium chloride treatment and a brief heat shock make E. coli take up plasmids. Only a small fraction of cells do.
- Selection: the plasmid carries an antibiotic-resistance gene, such as one for ampicillin resistance. On a plate containing the antibiotic, only cells that took up the plasmid survive and form colonies.
- Screening: some plasmids also carry a marker gene that the inserted DNA interrupts, so colonies with and without the insert look different.
| Plate | Cells | Expected growth |
|---|---|---|
| No ampicillin | Not given plasmid | A lawn (solid layer) |
| Ampicillin | Not given plasmid | None |
| No ampicillin | Given plasmid | A lawn |
| Ampicillin | Given plasmid | Separate colonies: the transformed cells only |
Key idea
In transformation, cells take up a plasmid; an antibiotic-resistance gene lets only transformed cells grow on selective plates. Sequencing reads the order of bases; in the chain-termination (Sanger) method, fragments that stop at each base are sorted by size, and the order of sizes gives the sequence.
Why it works
How does chain-termination sequencing work? Copies of the DNA are made with a few special nucleotides (dideoxynucleotides) that lack the 3′ –OH, so no more nucleotides can be added after one is built in (the 3′ rule again). Each copy stops at a random point, ending in a known base, often marked with a color. Sorted by length, the fragments spell out the new strand base by base from its 5′ end. The template is its complement.
Worked examples
Worked example: Transformation efficiency
Cells were given 0.1 µg of plasmid in 1.0 mL. A quarter of the mixture (0.25 mL) was spread on an ampicillin plate, and 120 colonies grew. What is the transformation efficiency?
- DNA on the plate: $0.1 \times 0.25 = 0.025$ µg.
- Efficiency = colonies ÷ µg of DNA spread: $120 / 0.025 = 4{,}800$.
Answer: 4,800 transformants per µg of DNA.
Worked example: Reading a chain-termination result
Fragments ending in each base were sorted by length: 1 nucleotide ends in G, 2 in A, 3 in T, 4 in T, 5 in C, 6 in A. What is the new strand, and what is the template?
- Shortest to longest gives the new strand from its 5′ end: G, A, T, T, C, A.
- New strand: 5′-GATTCA-3′.
- Template (complement, antiparallel): 3′-CTAAGT-5′.
Answer: New strand 5′-GATTCA-3′; template 3′-CTAAGT-5′ (5′-TGAATC-3′ written the standard way).
Common mistake
Thinking every cell on the plate took up the plasmid. On a plate without antibiotic, plasmid-free cells grow too. On an antibiotic plate, only transformed cells survive: the plate selects them, and each colony grew from one transformed cell. That is why efficiency counts colonies on the selective plate only.