07 OCT 2026 (RECONSTRUCTED)

To evaluate colony growth across experimental libraries, check the control strains (E. coli HB101 and DH5α + pAT64) from Oct 06, store the plates at 4°C and start fresh overnight cultures of the control strains.

• Incubated assay selection plates (Inoculated October 06)

• Lysogeny Broth (LB) liquid medium

• Tetracycline (tet) stock solution 

• Sterile glass test tubes

• Sterile 50 mL Falcon tube 

• Sterile Serological and micropipettes

• Sterile inoculation loops/sticks

• Retrieved all plates from the 37°C incubator after overnight incubation.

• Visually assessed all plates to look for colonies. Checked control plates side-by-side with experimental libraries to confirm the antibiotic plates were working.

• Placed all verified plates inside a sealed container and transferred them into the 4°C laboratory refrigerator to slow growth and keep the colonies for later experiments.

Inoculation of Overnight Cultures

• Working under aseptic conditions inside a fumehood, dispensed LB medium into a sterile 50 mL Falcon tube using a sterile serological pipette.

• Aseptically transferred 5 mL of the LB medium into each of the five clean glass test tubes using a fresh sterile serological pipette.

• Labelled the glass test tubes sequentially as follows:

  • Tube 1,2 : DH5α (pAT64) + tet

  • Tube 3,4 : HB101 (Empty)

  • Tube 5: Control (Negative Control)

• Added tetracycline (tet) 20 µg/mL to Tube 1 and 2 to maintain selective pressure for the pAT64 plasmid using a micropipette.

• Using a sterile inoculation loop, picked a single, well-isolated colony from the DH5α + pAT64 plate and submerged it into Tube 1 and then used a fresh loop for Tube 2.

• Using a fresh sterile loop, picked a single, well-isolated colony from the empty HB101 plate and submerged it into Tube 3 (no antibiotic) and then repeated this for Tube 4 with a fresh loop.

• For Tube 5 (Control), dipped an empty, unused sterile inoculation stick directly into the LB broth to check for environmental contamination.

• Placed all five glass tubes into the incubator shaker at 37°C shaking for overnight incubation (~12–16 hours).

Reading the plates

The controls are read first. If HB101 grows only on LB, and DH5α + pAT64 grows only on LB and Tet, then the plates and antibiotics behaved as expected, and any growth on the library plates can be put down to the DNA fragments those clones carry.

What this round showed?

Both controls gave the expected pattern, and library clones grew on IP and MP to different extents. The diluted libraries gave clearer, better-separated growth than undiluted ones, so dilution is worth keeping for future screens.

Why store at 4°C?

Cold storage slows growth, so colonies stay small and separate and can still be picked for re-streaking days later.

Next step

Repeat the screen with fresh cultures (Oct 08) to check that the result is reproducible.