Bacterial media

Bacterial media kdorfman Thu, 01/17/2013 - 18:53

Freezing bacteria

Freezing bacteria kdorfman Sat, 12/05/2015 - 18:44

To Freeze:

  • Pick a new colony, grow overnight in LB

  • inoculate LB from the overnight culture

  • Put 0.15 mL glycerol into sterile cryovial.

  • Add 0.85 mL mid-log culture (OD =~0.4). Pipette up and down to mix thoroughly

  • Freeze. Store in -80 if possible.

To thaw:

  • Scrape surface of frozen stock with sterile stick

  • Streak on agar plate

LB Broth

LB Broth kdorfman Fri, 09/25/2020 - 17:54

LB broth from mix

LB broth from mix kdorfman Tue, 05/28/2013 - 17:48

LB Broth Miller Luria-Bertani

Fisher DF0446-17-3 500 g $40.61.

25 g/L

To make mL LB broth
add: grams LB mix, stir till dissolved.
autoclave: minutes

LB from scratch

LB from scratch kdorfman Fri, 09/25/2020 - 17:38

LB (Miller) from scratch

To make mL LB broth from scratch
add: grams tryptone
add: grams yeast extract
add: grams NaCl
autoclave: minutes

LB agar

LB agar kdorfman Thu, 01/17/2013 - 18:55

LB agar granules

LB agar granules kdorfman Tue, 06/25/2019 - 20:01

Make with granulated LB (Miller) agar, Fisher BP9724

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
addg LB agar *granules*, stir till dissolved
autoclave for: minutes

LB agar powder

LB agar powder kdorfman Tue, 06/25/2019 - 20:00

Make with LB (Miller) Agar powder, Difco 244510

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add g LB agar, stir till dissolved
autoclave for: minutes

LB broth + agar

LB broth + agar kdorfman Tue, 06/25/2019 - 20:00

25 g LB + 15 g agar /liter

To make LB agar plates (100 mm)
you'll need: mL water
add: g LB broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

LB antibiotic

LB antibiotic kdorfman Wed, 10/01/2014 - 16:34

Table of antibiotic stability in poured agar

To make:
mL LB
with:
µg/mL antibiotic
and the antibiotic stock is:
mg/mL. Cool slightly.
then add:
µL antibiotic stock solution

line antibiotic
black Carbenicillin (ampicillin)
green IPTG

LB-sugars

LB-sugars kdorfman Wed, 10/01/2014 - 16:19

LB + sugars for Lac-operon work

sugar (or analog) MW
mono saccharide 180.2
disaccharide 342.3
IPTG 238.3

glucose, galactose, or fructose
To make mL LB broth
that is: M sugar,
add: g monosaccharide (glucose, galactose, or fructose)

lactose, maltose, sucrose
To make mL LB broth
that is: M sugar,
add: g disaccharide (lactose, maltose, sucrose)

Lactose is not very soluble in cold water.. Boil water, then set it to stir on a stir plate. Gradually tap in the lactose powder.

IPTG
To make mL LB broth
that is: mM IPTG,
add: µL 1M IPTG

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey agar

MacConkey agar kdorfman Thu, 01/17/2013 - 19:41

MacConkey Agar

  • Fisher 212122 2 kg (Difco)
  • Krackeler 10-211387 500g (via Sigma)
  • HiMedia MacConkey agar, granulated GM081 (Fisher NC1876763, VWR 95020-204)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

To make Maconkey agar plates
at mm plate diameter
pour mL per plate
start with: mL hot water
add g Maconkey agar, heat to boiling, stir till dissolved
autoclave for: minutes

leave stir bar in

put bottles on stir plate near sterile hood until handle-able

If you accidentally buy

Min med for GFP cells

Min med for GFP cells kdorfman Tue, 10/23/2018 - 15:53

From https://wahoo.cns.umass.edu/node/857

For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4

To make mL min med
start with mL 10X M9
add: mL 100% glycerol
add: mL 10mM FeSO4
Bring to final volume, filter sterilize

Mueller Hinton agar

Mueller Hinton agar kdorfman Thu, 01/17/2013 - 20:05

90922 Mueller Hinton Broth 2 from Sigma

22 grams per liter, consisting of:

Casein acid hydrolysate 17.5
Beef extract 3.0
Starch 1.5

Final pH 7.3 +/- 0.2 at 25°C

To make Mueller-Hinton plates (100 mm)
you'll need: mL water
add: g MH broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

P-Glo Bacterial media

P-Glo Bacterial media kdorfman Mon, 08/26/2019 - 22:26

Make 60 mm plates

2019 - for QBoC:

  • transformed pglo plasmid (1660405EDU) from the BioRad kit into
  • DH5 alpha E. coli (Invitrogen 18265-017) from the -80
  • Lyophilized cells (BioRad 1660408EDU) did not arrive in time

Used the Invitrogen protocol.

Plated on:

  • LB
  • LB amp
  • LB amp ara

Saw gfp colonies on the arabinose plates. Collected, grew up in LB amp, froze in 15% glycerol.

LB-Amp-Ara

LB-Amp-Ara kdorfman Mon, 08/26/2019 - 22:27

LB with Ampicilin or Carbenicilin and arabinose

Arabinose stock 200mg/mL in water, sterilized by filtration (=100 X)

Final concentration = 2 mg/mL

  • Arabinose MW = 150.13
  • 2 mg/mL = 2 g/L x 1 mol/150.13 g = 0.013M = 13 mM

Carbenicilin stock is 1000X

minimal medium (glycerol)

minimal medium (glycerol) bcrcstaff Wed, 11/14/2018 - 17:34

For motility strains

To make mL min med
start with mL 10X M9
add: mL 200X HMLTT
add: mL 100% glycerol
add: mL H2O
add: mL 1M MgSO4
add: uL 1M CaCl2, stir if it precipitates
Bring to final volume