Contributors: Esther, Chloe
Competent cell prep — second attempt
Inoculated a single colony (~5 µl) into 10 ml LB (Top10), cultured overnight at 37 °C, 200 rpm.
Chloramphenicol plates
Made chloramphenicol (40 mg/L) plates (~1 hour, prepared by Heather):
- Weighed 400 mg chloramphenicol powder, dissolved in 15 mL 100% ethanol
- Added 1 ml stock to LB agar, poured ~20–25 mL per plate
- Solidified at room temperature, stored sealed at 4 °C, protected from light
CxnA transformation
Transformed competent DH5α E. coli (from Heather’s freezer) with CxnA plasmid using standard heat-shock protocol (~3–4 hours):
- Thaw competent cells on ice 20–30 min
- Mix 5 µl plasmid DNA into 100 µl competent cells, incubate on ice 20–30 min
- Heat shock: 42 °C water bath, 45 sec
- Ice, 2 min
- Add 900 µl SOC media, grow 37 °C shaking, 45 min
- Plate 50–100 µl, incubate 37 °C overnight
Plated conditions: Chloramphenicol + His-tagged CxnA (growth); Chloramphenicol + CxnA (growth, ×2 plates); Ampicillin + His-tagged CxnA (small growth); Kanamycin + His-tagged CxnA (plated).
Label key: R = regular CxnA plasmid; H = His-tagged CxnA (backbone unknown).
Gel #1 — verify plasmid DNA and cut sites
Prepared 0.8% agarose gel (0.8 g agarose in 100 ml 1× TAE, melted, 5 µl gel red added once cooled). DNA samples from Chris. Run at 100 V for 1 hour.
Result: gel results not good — bands did not appear.