SBB11Ntbk-M.A.N.G.

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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):

  1. 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.
  2. Nikit and Marcus prepares 6 strips


Mon (4/11): Gary and Averee come in and perform large scale transformation: All stress promoters:GFP + one Con:GFP + 1 (-) control (36 transformations)

The negative control was plated onto its own round S/K plate.

Tuesday 4/12
Nikit picks colonies and we all plan our next move during class time.


for Tuesday (4/5)
  • 200µL of MC1061 competent cells
  • 1 toxR miniprep
  • spec plate

Procedure:

Transformation #1 (Marcus) - we need 1750 uL TSS mixture

  1. Thaw a 200 uL aliquot of MC1061 competent cells on ice
  2. Add 50uL of water
  3. Add 30 uL of KCM to the cells
  4. Put your toxR miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
  5. Add 70 uL of the cell cocktail to the miniprep,, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
  10. Plate 70+ uL on SPEC plate, let incubate at 37 degrees overnight

Preparing Competent Cells:

  1. Pick colony from Spec plate and grow in 3mL liquid media overnight (14-16 hrs.)
  2. Dilute 10% in fresh media. (Add 3mL culture in 27mL media flask) = 30mL solution
  3. Take OD measurements every half hour to make sure cells are in mid-log phase. ~ 2 hours.
  4. Do competent cell prep by adding 90uL of TSS for every mL of media left in the tube.
  5. Freeze flask?


Transformation #2

  1. Thaw a 1800 uL (200 uL ea) aliquot of MC1061/toxR competent cells on ice
  2. Add 450 uL (50 uL ea) of water to the cells (if greater volume is desired)
  3. Add 270 uL (30 uL ea) of KCM to the cells
  4. Put your miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
  5. Add 30 uL of the cell cocktail to the miniprep, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
  10. Plate 70+ uL on selective antibiotics on petri strips, let incubate at 37 degrees overnight
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