Results of Transformation of Kan and Tet Construction Plasmids
- Kan and Tet construction plasmid transformation did not succeed.
- No colony growth was observed on the Kan and Tet plates.
- Possible Reasons:
- May have been due to fewer colonies than usual being plated (300 µL, rather than 500 µL).
- May also have to do with the amount of DNA that we used - only 1 µL rather than 2 µL.
- Paul will bring over test plasmids that contain Kan and Tet resistance markers that we can use as a transformation control.
Results of Chloramphenicol Testing
- Growth observed in both LB Broth and LB Broth + Chloramphenicol tubes.
- This time round, chloramphenicol was either spread plated after cooling LB Agar, or added to LB broth at 1X concentration.
- Measured ODs by adding 2mL of bacterial culture into plastic cuvettes
- OD > 2 for both
| LB Broth + Chloramphenicol
| LB Broth Only
- Cuvettes were rinsed with tap water and returned to the cuvette storage styrofoam box.
- OD readings are bad as it means that either the cells are chloramphenicol resistant or that the method of preparation of chloramphenicol media isn't correct.
- Possible Causes:
- Concentration of chloramphenicol not high enough?
- Possibly need warmer temperatures for dissolving antibiotic into the media - but that may not be relevant as the dissolved antibiotic would just ppt out if it needed high temperatures for dissolution
- Will try using DH5alpha cells instead of DB3.1 cells to make sure it isn't the cells being resistant to current stock of chloramphenicol.
Testing of Chloramphenicol (yet again)
- Goal: The DB3.1 cells may be chloramphenicol resistant (not likely, but still it's a possibility).
- Logic here is that if DH5 alpha cells grow in the presence of chloramphenicol, then we know that the DB3.1 cells weren't the problem and that the chloramphenicol is the problem.
- So, test with DH5alpha cells to see if it's the antibiotic that doesn't work.
- Steps are as follows:
- Aliquoted remaining LB broth into 50 mL Falcon tubes.
- 10 mL LB Broth from one of the test tubes was pipetted into two test tubes each (i.e. each test tube contained 10 mL LB broth).
- 10 µL of 25 mg/mL stock Chloramphenicol was added to one of the test tubes, labeled as "LB + Chlor + DH5alpha". Other tube labeled as "LB Only + DH5alpha"
- Innoculated DH5alpha cells from glycerol freezer stocks into both test tubes.
- Incubated in Lagally Lab shaker at 37ºC overnight at 6:03PM.
- Poured 20 mL of molten LB Agar into each plate, cooled for 30 minutes
- 11 plates were spreaded with 20 µL of 100 mg/mL Ampicillin stocks, placed in AMBL 4ºC fridge in AMBL Prep Room
- 11 plates were stored as is (LB-only), also in AMBL 4ºC fridge in AMBL Prep Room.
- Remaining unused petri dishes were parafilmed in a sterile fashion and stored away in the Lagally Lab.
Transformation of GFP, J23100+RBS, pBAD and Terminator Biobricks
- To ensure that the late growth of the GFP, J23100+RBS, pBAD and terminator biobricks was not due to a degradation of the Ampicillin, a colony of each was replated on a new LB-Amp plate. All of the cells containing the biobrick parts grew on the new media.
- One colony from each of the GFP, J23100+RBS, pBAD and terminator plates were innoculated with 5mL of LB-Amp to be mini-prepped tomorrow