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

LB from scratch

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

LB (Miller) from scratch

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

LB agar powder

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

Make with LB (Miller) Agar powder, Difco 244510

LB broth + agar

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

25 g LB + 15 g agar /liter

LB antibiotic

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

Table of antibiotic stability in poured agar

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

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

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

(If you are going to make more than one bottle, turn on 65C waterbath in tissue culture room)

Turn on the stir-plate heat to 105C.

Withhold 50 mL of water to rinse the mouth of the bottle.

Heat the rest of the water to boiling in the bottle in the microwave.

Measure out the agar mix while the water is heating

Add the stir bar (CAREFULLY!) to the bottle of hot water

Put the bottle on the stir-plate

Add the agar mix to the bottle

Use the reserved water to rinse the granules stuck in the neck down to the liquid.

Stir and heat until granules are completely dissolved.

Cap loosely, wrap with foil, and autoclave.

(If you make more than one bottle, the extras go in the water bath till you are ready to pour)

leave stir bar in

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

If you accidentally buy

MacConkey + bile+ CV

MacConkey + bile+ CV kdorfman Mon, 01/08/2024 - 16:00

If you accidentally buy MacConkey without

bile salts (1.5 g/L)
Crystal violet (0.001g/L)
NaCl (5 g/L)

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

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

P-Glo Bacterial media

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

Make 60 mm plates

plain LB plates
LB/Amp (or Carb) plates (1 black stripe)
LB/Amp/Ara plates (1 black, 1 red stripe)

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.

Bacterial Transformation Lab Protocol.pdf

p-glo instructor manual

DH5alpha subcloning protocol

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

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

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

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

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

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

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

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

To make	mL min med
start with	mL 10X M9
add:	mL 100% glycerol
add:	mL 10mM FeSO4

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