Transformation results
- 73 transformants from last week's transformations
- Most from transformations with low cell density
- Most from transformations with 10 ul 1M sorbitol added prior to electroporation
- nothing shows at 14 hours or 24 hours
- Some colonies visible at 38 hours, more at 48, final at 60 or so
New standard transformation protocol
- culture cells in 45 ml 1161 medium to light yellow/slightly turbid
- Spin down and resuspend 3x with 45 ml EPB (no glycerol) on ice in chilled centrifuge
- Resuspend in 5 ml EPB on ice
- Aliquot 50 ul cells + 1 ul transposon DNA on ice
- Add 10 ul 1 M sorbitol
- Electroporate at 1.5 KV in 1 mm cuvette
- Immediately add 850 ul 1161 medium, place on ice
- Incubate 1 hour at 30C in the cuvette
- Plate 300 ul on 15 ug/ml TET plates
- Hold remainder in the cuvette for several days at 4C
Newly diluted transposase
- Dilute 50 ul transposase (8 ug/ul) into 800 ul transposase storage buffer
- Store at -20, label 500 ng/ul transposase
New transposomes
- 20 ul transposon DNA (83 ng/ul)
- 20 ul glycerol (use cut off tip for pipetting)
- 40 ul diluted transposase
- incubate 1 hour at 37C
- hold at -20C
New transformations
- standard protocol
- use 10 ul water, 5 ul water, no addition, 5 ul, 10 ul, and 15 ul sorbitol (1 M) additions
- Test for both positive and negative osmotic pressure changes, and narrower range than last time
- Plate out all of sorbitol added versions (3 plates)
- Plate out only 1 plate from water and no addition plates, save remainder at 4C
- Plates labeled with -10, -5, 0, +5, +10, +15 for water and sorbitol additions (ul added)
New plates and culture medium, standard protocol
Plans for genetics
- Promoters
- Reporters
- antibiotic
- recT recombination gene
- Test constitutive and heat shock promoters with reporter
- Test cat gene with constitutive promoter
- Combine heat shock promoter with recT gene
- Test recombination efficiency
|