SBB11Ntbk-M.A.N.G.
BE 140L Spring 2011 Team MANG
Marcus Macaulay
Averee Chang
Nikit Patel
Gary Dixon
Goal: Evaluate induction of stress by toxic genes in the promoter set for toxR. In essence, put Pbad-toxR in the cell, put a stress promoter-GFP in the cell, add arabinose until it starts getting toxic, measure the GFP on a plate reader (the Tecan).
Schedule
Tues (4/5): Marcus comes in at 9am transforms ToxR plasmid into MC1061 compitent cells. Plate on SPEC.
- Placed plated toxR cells in 37 degree C incubator labeled with M.A.N.G, date and time.
Wed (4/6): Nikit comes in at 9pm pick a colony and culture it in 3ml LB (tell GSI).
- Marcus went in at 15:10 and picked colonies.
- Labeled tube with red tape (BE 140L, Team M.A.N.G, date+time), placed in Warm Room (448A Stanley) in rack on the shaker closest to entry door on left side of room.
- Stored our plated colonies in class room lab's glass door refridgerator on 2nd rack from top, Labeled accordingly.
- See Picking Colonies for in-depth procedures as suggested by S. Boyarskiy.
Thur (4/7):
- Gary comes in at 9:30am. He dilutes the saturated 3ml culture into a flask full of 27ml LB. After 2 hours, performs small scale competence protocol. After resuspending in TSS (90ul x the number of starting ml of cells) freeze the cells in -80.
- Nikit and Marcus prepares 6 strips
Fri (4/7): Everybody comes in at class time and performs large scale transformation:
- All stress promoters:GFP and the one Con:GFP into ToxR cells (36 wells)
- All stress promoters:GFP into non ToxR cells (original) (35 wells)
- Constitutive promoter:GFP into non ToxR cells (original) (1 wells)
Things we have to tell/need from GSI:
- for Tuesday (4/5)
- 200µL of MC1061 competent cells
- 1 toxR miniprep
- spec plate
Procedure:
Transformation #1 (Marcus) - we need 1750 uL TSS mixture
- Thaw a 200 uL aliquot of MC1061 competent cells on ice
- Add 50uL of water
- Add 30 uL of KCM to the cells
- Put your toxR miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
- Add 70 uL of the cell cocktail to the miniprep,, stir to mix
- Let sit on ice for 10 min
- Heat shock for 90 seconds at 42 (longer incubation may work better)
- Put back on ice for 1 min
- Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
- Plate 70+ uL on SPEC plate, let incubate at 37 degrees overnight
Preparing Competent Cells:
- Pick colony from Spec plate and grow in 3mL liquid media overnight (14-16 hrs.)
- Dilute 10% in fresh media. (Add 3mL culture in 27mL media flask) = 30mL solution
- Take OD measurements every half hour to make sure cells are in mid-log phase. ~ 2 hours.
- Do competent cell prep by adding 90uL of TSS for every mL of media left in the tube.
- Freeze flask?
Transformation #2
- Thaw a 1800 uL aliquot of MC1061/toxR competent cells on ice
- Add 450 uL of water to the cells (if greater volume is desired)
- Add 270 uL of KCM to the cells
- Put your miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
- Add 70 uL of the cell cocktail to the miniprep, stir to mix
- Let sit on ice for 10 min
- Heat shock for 90 seconds at 42 (longer incubation may work better)
- Put back on ice for 1 min
- Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
- Plate 70+ uL on selective antibiotics on petri strips, let incubate at 37 degrees overnight