2011

2011 kdorfman Mon, 09/17/2012 - 17:55

E. coli for 3.1

E. coli for 3.1 kdorfman Wed, 10/12/2011 - 15:44

Streak GFP cells (from -80 freezer) on LB-Kan agar

Grow overnight culture in minimal medium + Kan (50 µg/mL).
(Aliquots in freezer are labeled in 1, 3, 6, 10 mg/mL.)
See here for Kan volumes

Each group needs 4 mL cells. Should prepare to have 8 mL/group = ~100 mL

  • Start 3 5 mL overnight cultures the night before.
  • Shake at 37C
  • Early morning: inoculate culture into minimal medium in flasks, turn off heat.
  • Throughout morning: Check OD's.
  • If OD > 0.8, dilute

  • If OD < 0.8, turn on heat

    OD should be ~ or just > 0.8 when students come for cells.

Students should get all their solutions ready before they come to get cells.

Dispense cells in glass tubes when students are ready.

For incubation with sugars, use the round bottom centrifuge tubes with the two stage caps from room 368. BD Falcon 352059. 14 mL polypropylene 17 x 100 mm, available at the Fisher stockroom. Fisher 14-959-11B.

During lab, spin the cells down in the big Eppendorf in 368. Don't turn on the refrigeration. Use the 6-tube adapters in the swinging buckets.

Media for Lab 3.1

Media for Lab 3.1 kdorfman Wed, 10/12/2011 - 15:50

Note: very small pellets, not too much green, except with IPTG. Dissapointing. Get a better pellet with 6 mL of culture + 4 minute spin (max speed = 500 with swinging bucket rotor). But still no obvious induction with lactose.

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

Adding Kanamycin to __mL medium

Kan mg/mL 10 25 50 100 250 500 1000 mL medium
1 0.5 1.25 2.5 5 12.5 25 50 uL Kan
3 0.17 0.42 0.83 1.67 4.17 8.33 16.67 uL
6 0.08 0.2 1 0.42 0.83 2.08 4.17 8.33 uL
10 0.05 0.125 0.25 0.5 1.25 2.5 5 uL

Sugar solutions

Ingredient vendor cat # MW mM 10 25 30 50 100 mL
glucose 180.15 500 0.9 2.25 2.7 4.5 9 g
galactose 180.15 500 0.9 2.25 2.7 4.5 9 g
maltose 342.3 500 1.8 4.5 5.4 9 18 g
lactose 342.3 500 1.8 4.5 5.4 9 19 g
IPTG (100mM) 0.4 0.004 0.01 0.012 0.2 0.4 mL

Each group needs

medium mL per group ~mL total
minimal 3 100
lactose 1 50
IPTG 0.75 25
gal 0.5 15
glu 1 30
malt 0.25 10

For Lab 3.2, each group needs enough as if to fill two rows of a plate with 100µL = 2.4 mL