Team:Montreal/Notebook/August

August 1

 * Punched spot of J40001 into 15uL (should be 5uL) of TE buffer then performed following transformations:

1. 6uL of J40001 into TOP10 cells with amp

2. 2uL of pUC19 into TOP10 with amp (control)

3. 6uL of pGFP into MC4100 calls with amp

4. 6uL of J40001 into dH5alpha cells with amp

August 2

 * Removed plates with transformations from previous day from incubator. Growth on all, except very minimal on dH5alpha cells with J40001.

August 4

 * Seeded 2 culture tubes of 5mL with

August 11

 * Transformed BL21 cells with T9002 from spot on plate 1009 11B soaked in 5uL of TE buffer. Plates (amp) were set in incubator.


 * Seeded

August 12

 * Growth observed on BL21 plates.


 * Prepared M9 Minimal media with 20mL of 20% w/v yeast broth, then autoclaved.


 * Seeded the T9002 plasmid in BL21 A1s in the following way:

all tubes contain 3mL of medium

1. Control M9+Yeast +dextrose with Amp

2. T9002 colony 1 in LB +Amp

3. T9002 colony 2 in M9/Yeast + dextrose +Amp

4. T9002 colony 1 in M9/Yeast + dextrose +Amp

@ 9:15 PM

Colony 1 is in two tubes: M9 and LB. If the M9 is bad we'll know because it will have grown in LB. If it doesn't grow in either then something's up.

August 13

 * Diluted culture #2 of T9002 in M9/Yeast in three tubes of 11x, 22x and 44x.

August 14

 * Spread 200uL of J40001 in BL21 cells from previous growth on two ampicillin-containing plates.
 * Seeded two starter cultures of T90002 in 7mL of LB with ampicillin at 9:37pm.

August 15
- 0.5mL of culture into 4.5mL of M9 minimal media with 20% yeast broth, then add 500uL of 5% dextrose solution. (11x) - 0.25mL of culture into 4.75mL of M9 minimal media with 20% yeast broth, then add 500uL of 5% dextrose solution (22x)
 * Dilute start colony of T90002 in 11x and 22x using the following:
 * After 1.5h, add 260uL of 10x AHL to 11X diluted solution and 130uL of 10x AHL to 22x diluted solution, then incubate for 1.5 more hours.
 * No observed fluorescence
 * Spun down 10mL of 10X diluted T90002 liquid colony, then add 400uL of 10X AHL. Incubate for 1 hour. No observed fluorescence.

August 18
- 2x 5mL LB culture seeded with J40001 in DH5alpha cells with ampicillin. - 2x 5mL LB culture seeded with T90002 in BL21 cells with ampicillin
 * Seed four starter colonies in 10mL culture tubes of the following:
 * Tomorrow, mini-prep followed by gel to be performed on all cultures to assess plasmid DNA identity.

August 19

 * Performed miniprep on all cultures (2x J40001 and 2x T90002)

August 20
- 2x 5mL LB culture seeded with J40001 in DH5alpha cells with ampicillin. - 2x 5mL LB culture seeded with T90002 in BL21 cells with ampicillin
 * Seed four starter colonies in 10mL culture tubes of the following:

August 21

 * Performed miniprep on all cultures (2x J40001 and 2x T90002)

August 22
- Yielded no visible bands under UV.
 * Gel electrophoresis on 08/19 and 08/21 miniprep DNA extractions.