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	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

2012

2012 kdorfman Mon, 09/17/2012 - 17:57

First thing, the morning before:

Pick a colony
Grow in 3 mL LB-Kan

Last thing, the day before: 100 µL culture into 40 mL (each w/ Kan)

LB
Lac 0.25 M
- 20 mL LB
- 20 mL Lac 0.5 M
IPTG 0.1mM
- 20 mL LB
- 20 mL 0.2 mM IPTG
Glu 0.25 M
- 20 mL LB
- 20 mL 0.5 M glu
Gal 0.25 M
- 20 mL LB
- 20 mL 0.5 mL gal
Mal 0.25 M
- 20 mL LB
- 20 mL 0.5 M mal
IPTG 0.1 mM + glu 0.25 M
- 20 mL 0.2 mM IPTG
- 20 mL 0.5 M glu
IPTG 0.1 mM + mal 0.125 M
- 20 mL 0.2 mM IPTG
- 10 mL LB
- 10 mL 0.5 M mal
Lac 0.25 M + glu 0.25 M
- 20 mL 0.5M lac
- 20 mL 0.5 M glu
Lac 0.25 M + mal 0.125 M
- 20 mL 0.5M lac
- 10 mL LB
- 10 mL 0.5 M mal
Gal 0.25 M + glu 0.25 M
- 20 mL 0.5 M gal
- 20 mL 0.5 M glu
Gal 0.25 M + mal 0.125 M
- 20 mL 0.5 M gal
- 10 mL LB
- 10 mL 0.5 M mal

Morning of:

Aliquot ~4 mL each culture into plastic tubes.
One set per 4 tables.
? save for next lab in fridge?

Media for Lac I

Media for Lac I kdorfman Mon, 09/17/2012 - 18:10

LB (150 mL)

LB +

0.1mM IPTG (60 mL)
0.5 M lac (60 mL)
0.5 M glu (80 mL)
0.5 M gal (60 mL)
0.5 M mal (40 mL)

Make enough for Lac operon I and II:

150 mL each + 50µg/mL Kan

lac = 27 g
gal = 13.5 g
mal = 27 g
IPTG = 0.3 mL 100mM stock

200 mL glu = 18 g

Kan calculation here:

https://wahoo.nsm.umass.edu/content/media-lab-31

2014

2014 kdorfman Fri, 10/03/2014 - 18:42

Mixtures needed to grow 50 mL cultures for qualitative experiment

Need an additional ~120 mL of each sugar for quantitative student experiments the next week. (24 5-mL aliquots each)

LB-sugar recipes

medium	LB	lac	IPTG	glu	gal	mal
a	50
b	25	25
c	25		25
d	25			25
e	25				25
f	25					25
g			25	25
h	12.5		25			12.5
i		25		25
j	12.5	25				12.5
k				25	25
l	12.5				25	12.5
Totals	212.5	75	75	100	75	62.5

Plus at least 100 mL for student experiments

To make 25 mL for each culture (6 sets of 12 4-mL cultures):

medium	LB	lac	IPTG	glu	gal	mal
a	25
b	12.5	12.5
c	12.5		12.5
d	12.5			12.5
e	12.5				12.5
f	12.5					12.5
g			12.5	12.5
h	12.5		12.5			12.5
i		12.5		12.5
j	6.25	12.5				6.25
k				12.5	12.5
l	6.25				12.5	6.25
Totals	106,25	37.5	37.5	50	37.5	31.25

2015

2015 kdorfman Thu, 10/08/2015 - 18:36

Make enough media for 3.1 (25 mL) and 3.2 (200 mL of LB, 150 Lac, 150 glu, 100 each of the others)

Calculate amount of each medium assuming they will make mixtures in 2 mL tubes. Each group needs ~12 mL. Mostly LB, then lac, then glu

for 3.1:

Each pair gets 1 set of 12 tubes, each with E. coli already grown up. LB +
- nothing
- lac (0.25 M)
- IPTG (0.01mM)
- glu (0.25 M)
- gal (0.25 M)
- mal (0.25 M)
- IPTG + glu
- IPTG + mal
- lac + glu
- lac + mal
- gal + glu
- gal + mal
Overgrown cultures all are too fluorescent!
Can use the same tubes for both lab sections (if students don't throw them out)
Night before: grow up GFP E coli from liquid culture in ~25 mL
morning of: aliquot cultures into tubes 1mL each.

Question of the day

Question of the day tfriedri Mon, 10/17/2011 - 20:55

Which chemical compounds influence lac operon expression?

Replace "concentrations" with "compound combination"

Replace "concentrations" with "compound combination" tfriedri Mon, 10/17/2011 - 20:59

Replace "concentrations" with "compound combination" in the question why they were chosen. Prevents answers like: To have a final vol. of 1 ml.

Or make it a specific question to be answered in the report. As it is nobody got the idea behind it.

3.1 Lac operon I