Chronological lab notes — June → October 2025
Preparation of kanamycin LB plates.
Transformation of pET28a.
Collection of plates.
Preparation of LB and SOB media.
Prepared starter plate. Streak plate.
Collection of starter plate.
cultivate NiCo21 and DH5α overnight culture
Harvest overnight culture of NiCo21 and DH5α
DH5α culture did not reach desired OD; NiCo21 overgrowth, all samples are not able to proceed to the next step
Reasons: SOB contamination; increased ambient temperature (deviated from usual 18°C); more vigorous shaking than expected.
PCR of the newly arrived gene fragments from IDT
Gel electrophoresis, smearing is found and will adjust the conditions in the coming days
PCR. Preparation of medium.
Competent cell preparation. Starter culture of DH5α showed a negative result. NiCo21 gives positive results.
Harvest overnight culture of NiCo21. Failed to proceed, the optical density in 600nm wavelength of the bacteria culture exceed target OD 0.55.
Preparation of materials: 800mL LB *2 (total 1.6L); 500mL conical flask *6; 100mL tube *2 box.
Gel electrophoresis of PCR products from 19/7.
DNA purification of same batch of PCR products.
PCR to test conditions for the 6 gene fragments. Gel electrophoresis of PCR products.
Preparation of overnight culture of NiCo21 and DH5α for competent cell preparation. Both culture showed positive results before the lab end on 23/7.
Harvest overnight culture of NiCo21 and DH5α.
Preparation of materials.
Preparation of overnight culture of NiCo21 and DH5α.
Harvest of cells. Perform DNA purification. Pick clones from the plate made in 26/6 and inoculate an overnight culture for plasmid preparation. Preparation of agar plates.
Plasmid preparation. Gel electrophoresis to check results. Restriction enzyme digestion.
Stock check.
DNA purification.
DNA purification.
Cultivation of overnight culture of NiCo21 and DH5α. PCR for the 6 fragments. DNA purification for the 6 fragment.
Gel electrophoresis of the PCR products from 7/8. Proceed with the preparation of competent cells, transparent solution, negative result obtained.
Cultivation of overnight culture.
Proceed with the preparation of competent cells, prepared 20 tubes of stock for each cells and stored in -80℃.
Transformation of pET28a.
Make Ligation product. Measure the concentration of the DNA in the plasmid. prepare plasmid RE.
Perform gel electrophoresis. Cut band.
Gel purification. Transformation of the ligation product to E. Coli.
Do gel electrophoresis. Check colonies in plates.
Transformation pET28a to DH5α. Dilute pET28a. Prepare agarose gel.
Pet28a transformation. Perform RE and ligation. Check stock of diluted pet28a. Dilute pet28a.
Prepare Kan working solution. Check clones of transformed pet28a —> no clones are presented (failed).
Check whether there are uncut MNEI, T1R2 and T1R3. Perform PCR. Prepare LB. Transformation with newly diluted pet28a.
Plasmid preparation. Check plasmid band. RE plasmid. PCR.
Gel electrophoresis of 16/9 product and RE product.
PCR Clean.
Transformation. Plasmid preparation. PCR of IDT DNA. Prepare agarose gel. Measure DNA concentration.
Prepare agarose gels. Make Kan plates. Measure plasmid concentration. Perform gel electrophoresis. Pick clone from the plates spreaded.
Plasmid RE using PCR machine.
Prepare agarose gels.
Perform gel electrophoresis on plasmid prep product on 18/9 (using 1 kbp ladder) and RE products (using 1k and 50bp) on 18/9. Prepare LB medium. Take out the overnight culture for plasmid prep. Prepare glycerol stock for pET28a. Plasmid purification. Elute the DNA from the plasmid prep. Measure the DNA concentration of the eluted plasmid.
Plasmid prep. Perform gel electrophoresis (plasmid prep products). Perform gel purification (plasmid prep products). Check DNA concentration of the plasmid prep. RE (1h elution incubation). Perform electrophoresis (RE) & Gel purification (RE). Ligation (using gel purification products).
Prepare gel for gel purification (0.6%). Perform gel electrophoresis for RE protocol. Switch the ligation condition from 4°C to 16°C (PCR machine) and continue incubation. Transformation of 1:3 & 1:5 Ligation product (MNEI Fragment 1-4, T1R2 & T1R3) to DH5a and NiCo 21 (Failed). Perform gel purification. Elution. Quantification of the DNA concentration. Ligation. Prepare 1L LB-agar. Make Kan plates. Perform plasmid prep. Elution. Perform gel electrophoresis on plasmid prep products. Check the concentration of the DNA in the plasmid prep products.
Prepare agarose gel.
Plasmid prep. Make Kan plates. Perform gel electrophoresis (RE product).
Gel purification (continue).
Transformation of 1:3 & 1:5 Ligation product (MNEI Fragment 1-4, T1R2 & T1R3) to DH5a and NiCo 21 (Again). Plasmid prep (failed since no bacteria appeared after centrifuge).
Make RE. Make Kan plate.
Pick clones of pET28a.
Perform gel purification. Measure DNA concentration of purified DNA concentration.
Pick clones of NiCo21.
Perform plasmid prep. Perform gel purification.
Make 2 gels (one for MNEIs, one for T1R2/3). PCR of the prepped plasmids and 2 RXN with just purified pET-28a. Plasmid prep for picked clones. Transform if the band size is correct. Perform gel electrophoresis.
Perform plasmid purification. Make 2 gels (2%). Measure the DNA concentration of transferred NiCo21 with 1:3 ligation products (MNEI Fragment 1).
Transform N1 to NiCo21 (failed since someone used PCR products of N1 to transfer to NiCo21). PCR for purified plasmids.
Prepare agarose gels. Perform gel electrophoresis for the PCR product.
Transform the N1 to NiCo21. Perform RE + Ligation (1:3).
Perform gel electrophoresis.
WIKI DONE