3.2: Lac operon II

3.2: Lac operon II kdorfman Tue, 10/18/2011 - 15:08

Quantitative Analysis of the Kinetics and Extent of Induction

Thursday 10/20/11 and Tuesday 10/25/11

2015_th_glowgrow.xlsx
2016_th_ps1.xls
2016_th_ps2.xls

E. coli for 3.2

E. coli for 3.2 kdorfman Tue, 10/18/2011 - 15:18

GFP E. coli grown in LB

Grow cells in LB-Kan 1

Overnight culture

Dilute and monitor in morning; keep cells in active growing phase. Turn off heat in shaker if necessary.

Need ~20 mL total (8x12x2x100uL)

OD of culture should be 0.4 when cells are added to plates. That will put the OD just down into the noise, so there will be a lag time at the beginning of the run.

TA aliquots 100 uL cells to student wells, so the OD in the well will be half of the OD in the culture.

100 µL in 96 wells on 2 plates = ~20 mL of cells

Run at RT to be comparable to growth curves (set thermostat to 25C).

Use Script Mode on the plate reader. Program is called E_coli_glow&grow.

Check layout - make sure all rows used are read!

Fix file names so OD, FL, ps1, ps2 are obvious.

Move all the files into a folder labeled with the lab day as soon as the program is through running - otherwise, the data files for the second lab day are interleaved with those for the first lab day. (Sorting by time created may help you out of this fix, though.)

The 12 minute pause is to make the cycle take 15 minutes. Check that lab manual explains this. Enter the time on the compiled file in 15 minute intervals.

  1. Consider growing cells in LB+glu, then spinning down and resuspending in LB. They are slightly fluorescent in LB. ↩︎

reagents for 3.2

reagents for 3.2 kdorfman Tue, 10/18/2011 - 15:24

2013 - run program for 12 hours. long enough to get past stasis, and show that fluorescence continues to increase.

Give each group of 4 5 mL of each medium

Let them mix in microfuge tubes if they want.

NOTE: plate lid got stuck on PS2 - data completely worthless. Try grinding down the bottom, different lids, tape. Do a dummy run in the morning.

Had to raise up the detector by putting slices of blue ruler under its frame. Also tape the lid to the plate, and the plate to the tray.

LB + Kanamycin (50 µg/mL)
Aliquots in freezer are labeled in 1, 3, 6, 10 mg/mL

If students loaded a whole row with a single solution, they would need 1.2 mL.

antibiotic calculator

Aliquot ~1.5 mL of each medium:

Totals for both days (25 groups)= 37.5 mL

Make 50 mL each
(If there are any left over from the previous year, make ~35 mL, because that is how much can be sterilized in one go with the 35 mL syringe + filter.)

Adding Kanamycin to __mL medium

Kan mg/mL 10 25 35 50 100 250 500 1000 mL medium
1 0.5 1.25 1.75 2.5 5 12.5 25 50 mL Kan
3 0.17 0.42 0.595 0.83 1.67 4.17 8.33 16.67 mL
6 0.08 0.21 0.252 0.42 0.83 2.08 4.17 8.33 mL
10 0.05 0.125 0.175 0.25 0.5 1.25 2.5 5 mL

Sugar solutions

Ingredient vendor cat # MW mM 35 50 100 250 mL
glucose 180.15 500 3.15 4.5 9 22.5 g
galactose 180.15 500 3.15 4.5 9 22.5 g
maltose 342.3 500 6.3 9 18 45 g
lactose* 342.3 500 6.3 9 18 45 g
IPTG (100mM) 0.1 0.035 0.05 0.1 .25 mL

Filter sterilize! Do not autoclave media with sugars!
* lactose takes a long time to go into solution. heat and stir overnight, covered with parafilm.