Recipes

Antibodies

For initial dilution of lyophilized antibody:

  • Dissolve to recommended concentration (per package insert) with half glycerol and half water.

  • Aliquot and freeze.

  • Label the tubes or the box with how much to dilute.

Primary

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Hisotology primary AB

mouse anti actin

DSHB JLA20

stock concentration = 22 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 90 uL ~11 mg/mL

Add 216 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti alkaline phosphatase

DSHB B4-78

stock concentration = 38 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~19ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

mouse anti myosin

DSHB T14

mouse anti-myosin light chain

stock solution: 37 ug/mL

working solution: 2 - 5 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~18ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

mouse anti synaptic vesicle

DSHB SV2

Synaptic vesicle glycoprotein 2A

Stock concentration = 36 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 60 uL ~18 mg/mL

Add 246 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti troponin

DSHB TI-4-s

Anti-troponin I

IgG (mouse)

Stock concentration = 43 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 50 uL ~22 mg/mL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti-collagen II

DSHB II-II6B3

stock solution: 96 ug/mL

working solution: 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 20 uL ~48 mg/mL

Add 286 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti-collagen IV

DSHB M3F7

mouse anti collagen IV (basement membranes)

2 tubes as of spring 2018:

  • stock concentration (8/7/08) = 30 ug/mL
  • stock concentration (12/22/11) = 39 ug/mL

working concentration = 2 - 5 ug/mL

Each diluted with glycerol to a concentration of 18.5 ug/mL

Aliquots are 50 uL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti-laminin

DSHB 2E8

mouse anti-laminin gamma 1

stock concentration = 26 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 80 uL ~12 mg/mL

Add 226 uL PBS-Tw-BSA to make enough for 3 slides

mouse anti-mucin

Monoclonal Anti-Mucin Gastric antibody produced in mouse

Sigma M5293

dilute 1:200 for use

dilute 1:1 in glycerol

Aliquot 4 uL; add 302 uL PBS BSA to make enough for 3 slides

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rabbit anti actin

Sigma A2668

Dilute 1:40 for use

Dilute 1:1 with glycerol

Aliquot 16 uL

Add 290 uL PBS BSA to make enough for 3 slides

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rabbit anti connexin

Anti-connexin-32 (265-279)

Sigma C3470

Dilute 1:600


Anti-Connexin-32 (106-124)

Sigma C3595

Dilute 1:400

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rabbit anti laminin

https://www.sigmaaldrich.com/catalog/product/sigma/l9393?lang=en&region=US

Sigma L9393

3 vials as of Spring 2018

dilute 1:25

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rabbit anti myosin

Sigma M7523

Formalin fixation

2 vials as of spring 2018

Dilute 1:20

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rabbit anti-neurofilament

Anti-Neurofilament 200 antibody produced in rabbit

Sigma N4142

Dilute 1:80

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rat anti connexin 32

DSHB R5.21C

stock concentration = 28 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 70 uL ~14 mg/mL

Add 236 uL PBS-Tw-BSA to make enough for 3 slides

rat anti integrin

DSHB Ralph 3.1

rat anti-integrin-3

IgG2a

stock concentration = 27 ug/mL

working concentration = 2 - 5 ug/mL

Histology secondary Ab

goat anti-mouse 488

Invitrogen A-11001

Ex: 499 nm

Em: 488 nm

Stock(as purchased) = 2 mg/mL

Stock bottle has been diluted with glycerol: ~800 uL of 1 mg/mL

Working concentration = 1 ug/mL

Aliquots are 1 uL of 1 mg/mL

Add 611 uL to make enough for 6 slides, or up to 1 mL

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goat anti-rabbit 546

Molecular Probes A-11010

Stock concentration = 2 mg/mL

Working concentration = 4 ug/mL

Em = 575 nm

Ex = 560 nm

ALL OUT as of Spring 2018

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goat anti-rabbit FITC

Goat anti-rabbit IgG FITC

Sigma F0382

Dilute 1:80

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Rabbit anti PKC

PKC alpha (c-20)

rabbit polyclonal Abs 1gG

DO NOT FREEZE

dilute 1:50 to use

Santa Cruz Biotechnology sc-208

From Wikipedia: Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.[5]

Protein kinase C-alpha (PKC-α) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKC-α has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit. Many studies are currently being conducted investigating the structure, function, and regulation of this enzyme. The most recent investigations concerning this enzyme include its general regulation, hepatic function, and cardiac function.

Secondary

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Bacterial media

Freezing bacteria

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 agar

mL medium mm plate diameter
10 60
25 100
52 150

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

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

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

LB antibiotic

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

LB broth

LB Broth Miller Luria-Bertani

Fisher DF0446-17-3 500 g $40.61.

25 g/L

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

LB-sugars

LB + sugars for Lac-operon work

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

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey agar

MacConkey Agar

  • Fisher 212122 2 kg (Difco)
  • Krackeler 10-211387 500g (via Sigma)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

To make MacConkey agar plates (100 mm)
you'll need: mL water
add: g MacConkey mix, heat & stir till dissolved
autoclave: minutes

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

leave stir bar in

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

Mueller Hinton agar

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

Buffers

Here are some commonly made buffers for biochemistry and molecular biology

HBSS

DEB

DNA Extraction Buffer for Gene & Genome

100 mM NaCl, 50 mM Tris, pH8, 25 mM EDTA, 1% SDS

Make from stock solutions

To make mL DNA Extraction Buffer
add: mL 5M NaCl
add: mL 1M Tris, pH8
add: mL 500mM EDTA
add: mL 20% SDS
add: uL BMe AT THE LAST MINUTE!

ingredient cf ci 100 200 mL
NaCl 100 mM 5000 mM 2 4 mL
Tris pH 8 50 mM 1000 mM 5 10 mL
EDTA 25 mM 500 mM 5 10 mL
SDS 1 % 20 % 5 10 mL
BME 10 mM 12564 mM 79.5 159 µL (at last minute!)

HBS (Ca-)

Calcium-free HBS

Make from dry ingredients:

To make mL Calcium-free HBS
start with g MgCl2
add: g KCl
add: g NaCl
add: g EGTA
add: g HEPES

Compound MW mM 1000 500 350 250 100 mL
MgCl2(6H2O) 203.3 0.493 0.1 0.05 0.035 0.025 0.01 g
KCl 74.56 2.67 0.2 0.1 0.07 0.05 0.005 g
NaCl 58.43 137.9 8 4 2.8 2 0.8 g
EGTA 380.4 0.1 0.038 0.0192 0.0134 0.0095 0.00384 g
HEPES 238.21 10 2.383 1.1915 0.858 0.596 0.238 g

raise pH to 7.3 with NaOH

1X HBS (ca-) from stock solutions

CHECK THIS

Ingredient stock M mM 0.25 0.5 1 2 L
MgCl.6H2O 1 0.49 0.0001 0.0002 0.0005 0.0010 mL
KCl 1 2.67 0.0007 0.0013 0.0027 0.0053 mL
NaCl 5 137.90 0.0069 0.0138 0.0276 0.0552 mL
HEPES 1 10 0. 025 0. 050 0. 100 0. 200 mL

pH 7.3 with NaOH

filter sterilize


HBS/Ca/Mg

Ingredient1 1X (mM) 10X (M)
CaCl2 0.9 0.009
MgCl2 0.493 0.00493
KCl 2.67 0.0267
NaCl 137.9 1.379
HEPES 10 0.1

  1. Concentrations from Lab 9, Bioimaging 2008, Dave Gross 

10X HBS (dry)

10X HBS/Ca/Mg from dry ingredients

To make mL 10X HBS/Ca/Mg
add: g CaCl2.2H20 (MW = 147.02)
add: g MgCl.6H2O (MW = 203.3)
add: g KCl (MW = 74.56)
add: g NaCl (MW = 58.43)
add: g HEPES (MW = 238.31)

pH 7.3 with NaOH

Filter sterilize

10X HBS (stocks)

10 X HBS from stock solutions

To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

1X HBS (stocks)

To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

HEPES

HEPES 1M

MW = 238.3

7.149 g / 30 mL

11.915 g / 50 mL

pH to 7.3 with NaOH pellets (~5 g/L)

Filter sterilize.

PBS

Make 1X PBS from 10X

100 mL 10X + 900 mL H2O


10X PBS from stock solutions

from Sigma ready-made 1X:

M ingredient
0.01 Na-K Phosphate
0.138 NaCl
0.0027 KCl
To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M KH2PO4
add: mL 1M Na2HPO4.7 H2O (MW = 268.07)

pH to 7.3 with NaOH

sterilize


10X PBS from dry ingredients (Carrie's recipe)

To make L 10X PBS
add: g NaCl (MW = 58.44)
add: g KCl (MW = 74.55)
add: g KH2PO4 (MW = 136.09)
add: g Na2HPO4.7 H2O

pH to 7.3 with NaOH

sterilize

from stock solutions

10X PBS from stock solutions

from Sigma ready-made 1X:

M (1X) M (10X) ingredient
0.01 0.10 Na-K Phosphate
0.138 1.38 NaCl
0.0027 0.027 KCl
calculated from Carrie's dry ingredients recipe:
To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M KH2PO4
add: mL 1M Na2HPO4.7 H2O (MW = 268.07)

pH to 7.3 with NaOH

sterilize


OR

To match Sigma recipe:

Make 1M Na2HPO4 (base); 1 M KH2PO4 (acid)

Mix to pH 7.3

(should be ~38.25 mL Na2HPO4, 11.5 mL KH2PO4) from the Sigma Buffer Reference Center

pH mL Na Phos dibasic mL Na Phos monobasic
7.2 36.0 14.0
7.4 40.5 9.5

Then

To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M K-Na Phosphate

PBS-Tw-Azide

PBS with:

  • 0.1% Tween 20
  • 0.2 g/L Na Azide (=0.02 %)

per Liter:

  • 100 mL 10x PBS
  • 10 mL 10% Tween 20
  • 2 mL 10% Na azide
PBS-Tw-Az: L
10X PBS: mL
10% Tween20: mL
10% Na Azide: mL

PIPES

0.5 M pH 6.7

(for transformation buffer for genetics)

MW (disodium salt) = 346.32

173.16 g/L

17.316 g/ 100 mL

Mix in ~80% of final volume

pH to 6.7 with 5M KOH

Filter sterilize

Aliquot

Freeze

Potassium phosphate

1 M Potassium phosphate buffer

To make 100 mL of 1M potassium phosphate:

pH mL 1M K2HPO4 mL KH2PO4 (mL)
5.8 8.5 91.5
6.0 13.2 86.8
6.2 19.2 80.8
6.4 27.8 72.2
6.6 38.1 61.9
6.8 49.7 50.3
7.0 61.5 38.5
7.2 71.7 28.3
7.4 80.2 19.8
7.6 86.6 13.4
7.8 90.8 9.2
8.0 94.0 6.0

from http://ivaan.com/protocols/151.html

To make approximately: mL Potassium phosphate buffer
with: pH
start with approximately: mL K2 (base)
and: mL K1 (acid)

Check the pH with the meter.

Add more K2 to raise the pH, or K1 to lower it.

Dilute as necessary to achieve the desired concentration.

Sodium Phosphate

To make 100 mL 1M sodium phosphate at a given pH:

pH 1 M Na2HPO4 1 M NaH2PO4
8.0 93.2 ml 6.8 ml
7.8 89.6 ml 10.4 ml
7.6 84.5 ml 15.5 ml
7.4 77.4 ml 22.6 ml
7.2 68.4 ml 31.6 ml
7.0 57.7 ml 42.3 ml
6.8 46.3 ml 53.7 ml
6.6 35.2 ml 64.8 ml
6.4 25.5 ml 74.5 ml
6.2 17.8 ml 82.2 ml
6.0 12.0 ml 88.0 ml
5.8 7.9 ml 92.1 ml

Dilute to the desired concentration.

T10E1

Tris 10 mM, EDTA 1 mM, pH 8

Make from stock solutions

To make mL T10E1
add: mL 1M Tris pH 8
add: mL 0.5M EDTA

ingredient cf ci 25 50 mL
Tris pH 8 10 mM 1000 mM 0.25 0.5 mL
EDTA 1 mM 500 mM 0.05 0.1 mL

T10E5

Tris 10 mM, EDTA 5 mM, pH 8

Make from stock solutions

To make mL T10E5
add: mL 1M Tris pH 8
add: mL 0.5M EDTA

ingredient cf ci 25 50 mL
Tris pH 8 10 mM 1000 mM 0.25 0.5 mL
EDTA 5 mM 500 mM 0.25 0.5 mL

TAE

Tris-Acetate-EDTA buffer for agarose gel electrophoresis

1X TAE

To make L 1X TAE
add: mL 50X TAE

plus water to final volume

50X TAE stock

from Maniotis

To make mL 50X TAE
add: g Tris base
add: mL glacial acetic acid
add: mL 0.5M EDTA
OR add: g EDTA

pH to 8 with acetic acid or NaOH

should this be 8.4?

Ingredient cf (mM) 1000 750 600 500 mL
Tris 2000 242 181.5 145.2 121 g
Acetic acid 1000 57.1 42.9 34.3 28.6 mL
EDTA 0.5 M 50 100 75 60 50 mL
OR
EDTA 50 18.62 13.965 11.172 9.31 g

pH to 8 with acetic acid or NaOH

Maniotis says to autoclave, but the salt concentration is so high that nothing will grow in it if you don't.

Taq dilution buffer

To dilute Takara Ex-Taq

Store in freezer. Won’t freeze solid. Should keep forever.

Cell culture media

FBS aliquots

FBS comes in 500 mL bottles.

Aliquot:

volume (mL) # aliquots for
50 3 500 mL DMEM or non-CO2 medium
37.5 4 500 mL F10-Ham's
25 4 250 mL DMEM or non-CO2 medium
18.75 4 250 mL F10 Ham's
6.25 4 to make freezing media

2013: Try

  • Fisherbrand™ Research Grade Fetal Bovine Serum
  • 03-600-511
  • 500 mL for $133

Krackeler 45-F0926-500ML $145

Carrie's brand: Atlanta Biologicals Premium S11150 ~$300

Also use Krackeler 12103C ~$314

Antibiootic/Antimycotic

Anti-Anti comes in 100 mL bottles.

Aliquot:

volume (mL) # aliquots for
10 1 1 L
5 12 500 mL
2.5 12 250 mL

Keep frozen until ready to use.

Fisher SV30079.01, $20

DMEM

Medium for 3t3 fibroblasts and B16 cells

To make mL DMEM
start with mL water
add: g DMEM*
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

  • if using DMEM without pyruvate, add 1 mL Na Pyruvate (100mM) per 100 mL medium
Ingredient supplier cat # 1 L 500 mL 250 mL
DMEM 13.4 6.7 3.35 g
NaHCO3 3.7 1.85 0.925 g
HEPES 1.3 0.65 0.325 g
FBS Krackeler 12103C 100 50 25 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix DMEM, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.5
Adjust pH to 7.2 with HCl (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

F10-Ham's

for all LLCPk cell lines

To make mL F10-Ham's
start with mL water
add: g F10 (Hams) (Sigma N6635)
add: g Optimem (Invitrogen 226000-050)
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1 L 500 mL 250 mL
F10 (Ham's) Sigma N6635 4.9 2.45 1.225 g
Optimem Invitrogen 22600-050 6.8 3.4 1.7 g
NaHCO3 1.8 0.9 0.45 g
HEPES 0.66 0.33 0.165 g
FBS Krackeler 12103C 75 37.5 18.75 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix F10, Optimem, NaHCO3, HEPES in about 70% of the final volume of dH2O.

Initial pH ~7.1
Adjust pH to 7.2 (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Refrigerate

For serum free, replace serum with distilled water

Freezing media

Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.

Filter sterilize

Non-CO2 Media

Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammali...

FluoroBrite™ DMEM Media

serum-free

Non-CO2 serum-free medium

For working with live cells at the microscope

To make mL non CO2 medium
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1000 500 250 mL
MEM Sigma M3024-1L 13.4 6.7 3.35 g
HEPES Acros 172571000 1.3 0.65 0.325 g
Na pyruvate 100 mM Thermo SH30239.01 10 5 2.5 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix MEM and HEPES in about 70% of the final volume of dH2O.

Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

with serum

Non-CO2 Medium

For working with live cells at the microscope, when serum is needed for normal division.

To make mL F10-Ham's
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1 L 500 mL 250 mL
MEM 13.4 6.7 3.35 g
HEPES 1.3 0.65 0.325 g
Na pyruvate 100 mM 10 5 2.5 mL
FBS Krackeler 12103C 100 50 25 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix MEM and HEPES in about 70% of the final volume of dH2O.

Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of FBS, antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

Trypsin

Try Fisher 12-605-010

Stable at room temp!

Gibco™ TrypLE Express Enzyme (1X), Phenol Red

Animal origin-free, recombinant enzyme

$20.10 -for 100 mL

Culture Media

Worm media

Freezing medium

Per 1 L

  • 100 mL 10X M9 salts
  • 240 mL glycerol
  • 300 µL 1M MgSO4
  • water to 1L

Filter sterilize

M9 for worms

Per 1 L:

  • 100 mL 10X M salts
  • 300 µL 1M MgSO4
  • water to 1 L

Filter sterilize

NGM

Per Liter of medium (~75 plates):

  • 975 mL Water
  • 3 g NaCl
  • 2.5 g Peptone (Fisher BP1420-500 $78.80)
  • 17 g Bactoagar

Autoclave with stir bar inside

Cool to 55C in a 55C water bath

Add per L (see recipes in stock solutions):

  • 1 mL cholesterol (5 mg/mL in 95% EtOH)
  • 1 mL CaCl2 (1 M, STERILE)
  • 1 mL MgSO4 (1 M, STERILE)
  • 25 mL K-phosphate buffer (1M, pH 6.0, STERILE1)

Swirl flask to mix

Dispense 10 mL into each 60mm dish.

Stack 10 high

Let stand for ~48 hours for condensation to evaporate

Pack in sterilized plastic boxes.

To make NGM agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone, stir till dissolved
addg NaCL, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add: mL 5mg/mL cholesterol
add: mL sterile 1M CaCl2
add: mL sterile 1M MgSo4
add: mL sterile 1M K-Phos buffer, pH 6

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


  1. 3.3 mL K2HPO4 + 21.7 mL KH2PO4 

Dilutions

Enter initial and final concentration (in matching units!), and final volume

c1 = stock concentration
c2 =concentration you're trying to make
v2 =volume you're trying to make
v1= Start with this volume, and add water (or other diluent) to final volume

Fixatives

How to fix cells and tissues

Formaldehyde fix

3.7% formaldehyde
0.5% Triton X
in PBS

Formaldehyde stock: 37%
Triton X stock: 10%

To make mL fixative
add: mL 37% formaldehyde
add: mL 10X PBS
and: mL 10% Triton-X

Methanol fix

100% methanol at ice temperature

10 min

No permeabilization step needed.

Wash with PBS afterwards.

The methanol fixation is an easy method; however, it frequently solubilizes and removes membrane bound antigens. By a simple precipitation of the protein, methanol only provides low structural preservation.

Paraformaldehyde Fix

3.7% paraformaldehyde
in PBS

Paraformaldehyde stock: 37%
Triton X stock: 10%

To make mL fixative
add: mL 37% paraformaldehyde
add: mL 10X PBS

Paraglut

3.2% paraformaldehyde
0.1% glutaraldehyde
0.5% Triton X-100

in PBS

To make mL fixative
add: mL 37% paraformaldehyde
add: mL 10X PBS
and: mL 10% Triton-X
add: mL 1glutaraldehyde

Bring to final volume with distilled water.

Gels

gels:
%:
TAE: mL
agarose: g
EtBr: µL

*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)

*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER

*Allow to cool slightly

*Add EtBr (in the fume hood)

*Aliquot into 50 mL conical tubes

*Put in rack in 65C waterbath

Hormones & Inhibitors, etc.

By compound

ATP

10 mM ATP Stock
(from Maniotis)

  • 60 mg ATP in 8 mL H2O
  • pH to 7.0 with 0.1 M NaOH
  • Bring volume to 10 mL with H2O
  • Aliquot and freeze.

Angiotensin

Sigma A9525 1mg/mL in water

Vasoconstrictor

Soluble in water 25 mg/mL (only use sterile water!)

MW: 1046.18 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Subject to degradation in freezer storage - wear gloves!

Make 1 mg/mL stock (= 1.0462 mM)

Working concentration 1 µM

Dissolve 1:1000 in HBS

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Blebbistatin

Myosin inhibitor

(-)-Blebbistatin Sigma B05650-1mg

Info from Cayman:

A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.

(±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.

(±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.

Note green fluorescent crystals seen in a 75µM solution.

Stock was 1 mg/mL in DMSO

44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium

REDO

1 mg in 34 µL DMSO = ~100mM

MW = 292

Use at ~150 µM

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Bradykinin

  • 9-amino acid peptide chain: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Bradykinin raises internal calcium levels

*Sigma B3259 - 1 mg septum bottle

  • MW = 1060.2

    1 mg/mL stock
    (= 1.0602 mM)

Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)

Aliquots are 21.2 µL.

2 µM solution for use
(=2.12 µg/mL)

Cannabinol

Tetrahydrocannabinol solution 1 mg/mL in methanol

Sigma T464

store at 4C

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Concanavalin A

  • Sigma C5275 5 mg
  • Plant mitogen
  • Lectin (carbohydrate-binding protein) extracted from the jack-bean, Canavalia ensiformis
  • Con A binds specifically to α-Mannose, α-Galactose structures found in sugars, glycoproteins and glycolipids

Concanavalin-coated coverslips

  • Make stock 5 mg/mL solution (add 1 mL sterile PBS to bottle – gently rotate to mix)
  • Keep sterile - work in sterile hood
  • aliquot 100 µL into microfuge tubes (cannot take repeated freeze-thaw cycles)
  • Coat coverslip bottom dishes with
    • 0.75 mL 0.1 mg/mL: 0.015 mL (5 mg/mL) + 0.735 mL PBS
    • 0.75 mL 0.5 mg/mL: 0.075 mL (5 mg/mL) + 0.675 mL PBS
  • Incubate the coverslip dishes for 1 hour 37˚C
  • Remove the solution
  • rinse in PBS
  • Air dry the coverslip.
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Cyclin Inhibitor

CKD1 inhibitor IV RO3306

1 mM in DMSO

A cyclin-dependent kinase inhibitor protein inhibits cyclin-dependent kinase. Several function as tumor suppressor proteins. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the G1 phase.

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Cytochalasin D

MW = 507.62
Sigma C2618: 200 µL 5 mg/mL (~10 mM) in DMSO
Sigma C8273: 5 mg. Dissolve in 1 mL DMSO

  • alkaloid mycotoxin produced by Helminthosporium and other molds.
  • disrupts actin microfilaments
  • binds to F-actin polymer
  • prevents polymerization of actin monomers

  • Aliquot 10 µL 10 mM

  • Store in the dark, in the freezer
  • Adding 490 µL of non-CO2 medium makes 500 µL of 200 µM
  • Final dilution should contain no more than 0.1% DMSO

  • 250 nM in the dish should give an effect

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Fibronectin

Sigma F1141

Attachment factor

Importazole

401105-10mg Calbiochem

Importazole is an inhibitor of importin-β transport receptors.

During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. Importazole somehow disrupts the importin/RAN interaction. Importazole is selective among other transporters. Compounds are imported into, but not out of, cells.

A cell-permeable diaminoquinazoline compound that selectively blocks importin-β-mediated nuclear import of NLS bearing cargos in a reversible manner.

Soluble in DMSO (25 mg/ml; clear, colorless solution)

5 mg/mL ailquots in freezer

Ionomycin

Raises intracellular calcium levels

Fisher or Invitrogen (Life Technologies) I24222

1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL

24 µL in 8 mL, aliquot 800 µL

They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
45 µL in 15 mL

Takes a long time to thaw. Vortex.

Aliquot 1.55 mL so they can do two 750 µL experiments

Jasplakinolide

(out of stock 2018)

  • Jasplakinolide – Santa Cruz biochemical, product sc202191 F
    • inducer of actin polymerization & stabilization; inhibits actin filament disassembly
    • F-actin probe
    • MW = ~710
    • powder is stable frozen over a year
    • stock is stable 3-4 mo at -20C
    • Make 1 mM stock with 50 µg in 70 µL DMSO
    • Add 5 mL non-CO2 medium to make ~14 µM
    • 50 nM to 5 μM working range
    • minutes to hours incubation time

LDL

Low-density lipoprotein from human plasma, Dil complex (Dil LDL)

1 mg/mL

200 uL

DO NOT FREEZE

Life Technologies L3482

Laminin

Laminin from mouse BD354232

1 mL 1 mM

Cell adhesion

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Latrunculin

Alexis Biochemicals 350-036-C100 Catalog # t110

Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes

Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.

Inactivated by FBS. (Rinse medium off before treatment.)

MW = 395.5

Soluble in DMSO and ethanol.

Store in freezer.

Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM

(2.5 µM = 25 x 100 nm)

Stock is 1mm

µL 1mM stock µL serum-free buffer or medium µM final concentration
1 * 99 10
1 399 2.5
1 999 1

*Use this to make further dilutions

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ML-7

ML-7 Sigma, 12764.

selective myosin light chain kinase inhibitor

  • soluble 10mg/mL in 50% EtOH
  • MW = 452.74
  • 5 mg in bottle
  • add 1100 µL to make 10 mM solution
  • used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
  • 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
  • make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM
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Melanocyte Stimulating Hormone

Sigma M4135 1 mg

Molecular Weight 1664.88

From Sigma: Amino Acid Sequence:

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

Pituitary hormone which causes darkening skin pigmentation from amphibians to humans. In mammals, it can also have behavioral effects on learning, attention, and memory.

Hormone that stimulates melanogenesis; facilitates learning and memory; affects inflammatory and immune responses and peripheral nerve regeneration.

α-Melanocyte-stimulating hormone (α-MSH) acts as an anti-inflammatory agent via down regulating the production and activity of the pro-inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF)-α and IL-6 expressed in various cells of the immune system. It also controls the nitric oxide production associated with inflammation. α−MSH inhibits nuclear factor-κB (NF-κB)-dependent gene transcription and NF-κB pathway induced by TNF and other inflammatory agents. This activity of α-MSH is mediated through the production of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) enzyme. α–MSH functions as a potent therapeutics for various conditions resulted through NF-κB activation including, inflammatory diseases, human immunodeficiency virus (HIV) replication in AIDS (acquired immunodeficiency syndrome), and septic shock.[2] α-MSH has an essential role to play in melanin production in animals. α-MSH regulates development of several skin diseases, including cutaneous inflammation and hyper-proliferative skin diseases.[3] Linkage

Derived from ACTH 1-13 Other Notes

Lyophilized from 0.1% TFA in H2O Packaging

1, 5 mg in glass bottle Application

α-Melanocyte stimulating hormone (α–MSH) has been used for following studies: • it has been Intracerebroventricularly (icv) injected to mice for behavioral studies.[1] • to determine the effect of α−MSH on growth of stationary Nb 2 node lymphoma cell cultures.[4] • to determine the effect of α−MSH on leptin secretion in the primary cultures of differentiated adipocytes.[5] • α−MSH promoted melanin production in the B16-F1 cells from murine melanoma cell line.[6] General description

α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide, mostly produced by the cells in the brain, pituitary and circulation.[2] Pro-inflammatory cytokines or UV light induced epidermal cells such as keratinocytes and melanocytes synthesize and discharge α–MSH. Poopiomelanocortin (POMC) acts as a precursor for α-Melanocyte-stimulating hormone (α-MSH) production.[3]

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Monastrol

Inhibits kinesin 5

200 mM in DMSO

Final concentration 200 uM

5.5 uL aliquots

Monensin

Monensin sodium salt

Sigma M5273

soluble in methanol 50 mg/mL

insoluble in water

soluble in DMSO, ethanol

ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells

inhibits transition from G1 to S

antiprotozoal, antibacterial, or antifungal agent

MW 692.9

1 µM = half-maximal inhibitory effect

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Nerve Growth Factor

Nerve Growth Factor 2.5S

Harlan.com Cat no. 5025

From Sigma: 2.5S subunit of nerve growth factor-7S (NGF-7S) is essentially the β-subunit when isolated from male mouse submaxillary glands under initially dissociative conditions by a modification of the method of Bocchini and Angeletti.

Stored at 4C

Nocodazole

Prevents polymerization of microtubules. Prevents cells from entering metaphase.

*Acros 358240100, 10 mg

*Soluble in DMSO

*Dilute stock in medium to treat cells.

*Make a 10 mg/mL (=33mM) stock: Add 1 mL DMSO to 10 mg in bottle.

  • Aliquot 3 µL 33 mM per tube.

*Working concentration range is usually 100nM - 30 µM.

  • add 97 µL to tube to make 100 µL of 1 mM

  • 33 µL 1mM + 10 mL medium = 3.3 µM

Poly-L-Lysine

Non-specific attachment factor for cells; use it to coat coverslips.

Sigma P1524

  1. Add 50 ml of sterile tissue culture grade water to 5 mg of poly-lysine.
  2. Aseptically coat culture surface with 1 ml per 25 cm2 of solution. Rock gently to ensure even coating of the culture surface.
  3. After 5 minutes, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
  4. Allow to dry at least two hours before introducing cells and medium. If glassware or slides must be sterilized after coating with poly-lysine, γ-irradiation is recommended instead of autoclaving.

Can also buy poly-lysine coated slides: Fisher 6776215

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STLC

  • STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

(+)-S-Trityl-L-cysteine is a cell-permeable selective inhibitor of mitotic kinesin Eg5 and ATPase activities.

KIF11 (also known as kinesin-5 and Eg5) is a homotetramer which cross-links anti-parallel microtubules in the mitotic spindle to maintain spindle bipolarity. The motor domain or motor head is at the N-terminus and performs ATP hydrolysis and binds to microtubules. (https://en.wikipedia.org/wiki/Kinesin_family_member_11)

  • Make 100 mM stock:

    • 1 g in bottle.
    • Mix with 27.5 mL DMSO
    • heat to 65C (~10 min), vortex
    • if necessary, filter sterilize to remove insoluble particles.
    • save as 1 mL aliquots in freezer. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)
  • Make 1 mM stock from the 100 mM stock

    • 10 µL 1mM stock
    • 990 µL DMSO
    • vortex
    • it will crystallize in the refrigerator. Warm and vortex to redissolve it.
  • Make 10 µL aliquots of 1 mM stock. Freeze

    • label says to add 990 µL medium to make 10 µM working solution
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Taxol

Taxol (20 µM final)

Fisher 109710 Paclitaxel.
Sigma T7402 1mg
MW = 853.91

  • Stabilizes the microtubule polymer
  • Protects microtulues from disassembly.
  • Prevents metaphase spindle configuration
  • Blocks progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the G-phase of the cell cycle without cell division.

  • soluble in DMSO, not water.

  • Add 0.117 mL DMSO to make a 0.01M (=10 mM) stock solution from the 1 mg powder

  • Aliquot 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)

    • add 490 µL to make 200µM
    • dilute into medium from there
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Trichostatin A

Cayman 89730

10 mM

Histone deacetylase Inhibitor

Trichostatin A (TSA), an antifungal antibiotic produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of histone deacetylases (HDACs), which are overexpressed in various cancers and closely correlate with oncogenic factors.

Trichostatin A is active at nanomolar concentrations in mammalian cells. By suppressing the activity of HDACs, it leads to increased histone acetylation, thereby causing highly acetylated histones to accumulate in the cell [2]. This in turn induces enhanced expression of specific genes that elicit extensive cellular morphologic and metabolic changes such as growth arrest, differentiation and apoptosis. At submicromolar concentrations Trichostatin A has been shown to induce apoptosis in diverse cancer cells while exhibiting very low toxicity to normal cells.

TSA is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, TSA should be directly dissolved in 0.1 M HCl (1.3 mg/ml) and then neutralized with PBS (pH 7.2) to achieve the desired concentration or pH. Approximately 0.7 ml of PBS (pH 7.2) is required to neutralize 1 ml of the acidic solution. We do not recommend storing the aqueous solution for more than one day.

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Valinomycin

Potassium Ionophore, MW: 1111.32

Sigma V0627

Insoluble in water

stock is 10 mg/mL in DMSO

  • cyclododecadepsi-peptide ionophore antibiotic.
  • potassium ionophore that transports K+ across biological and artificial lipid membranes.
  • can induce K conductivity in cell membranes
  • uncouples oxidative phosphorylation,
  • induces apoptosis in murine thymocytes, and in pre-B cell.
  • inhibits NGF-induced neuronal differentiation
  • antagonizes ET-induced vasoconstriction.
  • Useful in studies of K transport in mitochondria.
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Vasopressin

(=Anti-diuretic hormone)

causes calcium oscillations

1 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock

1 µL aliquots in freezer


1 µM dilution to make student solutions

1 µL 1 mM in 999 µL water

(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

By target

Target or Effect Drug(s)
Vasoconstriction Angiotensin
Non-muscle myosin II ATPase Blebbistatin
Internal calcium levels Bradykinin
Nervous system Cannabinol
α-Mannose, α-Galactose sugars, glycoproteins and glycolipids Concanavalin A
cyclin-dependent kinase cyclin inhibitor
actin polymerization cytochalasin D
cell adhesion fibronectin

fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL fully qualified URL

Naegleria medium

from dry

DIW (pure deionized water) 800 ml

K2HPO4 (anhydrous) 0.72 g

KH2PO4 (anhydrous) 2.16 g

Difco Bacto Peptone (not Proteose Peptone) 3.2 g

Difco Bacto Agar 12.0 g

Mix by swirling (no need to dissolve). Autoclave 10 min. Cool to 45–50° in a water bath, and pour plates about ½ full.

To make plates (100 mm)
you'll need: mL water
add: g K2HPO4
add: g KH2PO4
add: g peptone (Difco Bacto Peptone, not proteose)
add: g agar
autoclave for: minutes

from stock solutions

DIW (pure deionized water) 780 ml

1M K2HPO4 (dibasic) 4 mL

1M KH2PO4 (monobasic) 16 mL

Difco Bacto Peptone (not Proteose Peptone) 3.2 g

Difco Bacto Agar 12.0 g

Mix by swirling (no need to dissolve). Autoclave 10 min. Cool to 45–50° in a water bath, and pour plates about ½ full.

To make plates (100 mm)
(for a final volume of mL of medium)
start with: mL water
add: mL 1M K2HPO4
add: mL 1M KH2PO4
add: g peptone (Difco Bacto Peptone, not proteose)
add: g agar
autoclave for: minutes

Plant growth media

10 g agar per L

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LPGM

Lilly pollen growth medium

205 mM (7%) sucrose (See Sugars.)

1.6 mM H3BO3

0.1 mM CaCl2

15 mM MES, pH 5.7 with KOH

To make mL LPGM
add: mL 150 mM MES
add: uL 10 mM CaCl2
add: uL 160 mM Borate

Stock solutions:

150 mM MES

10 mM CaCl2

160 mM Borate

? 35 % Sucrose (filter sterilze)

MS high salt

150 too high - try 100mM for F 2015

150 mM NaCl

10 g agar/L

Make regular MS, then add 0.03 mL 5M NaCl per mL medium

100 mM NaCl

0.02 mL 5M NaCl per mL medium

MS iron-free

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

10 g agar/L

To make plates low-iron MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

MS low iron

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

To make plates low-iron MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL ferrous sulfate 10 mM
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

MS medium

To make 30 mL plates
add: mL water initial volume*
add: mL 10X macronutrients [1]
add: mL 10X micronutrients [2]
add: g MES. pH to 5.7 w/ 1M KOH**
bring volume to: mL water final volume
add: g bacto or phyto agar
autoclave for: minutes

*Add the other salt mixtures to the water to prevent precipitation

**pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs ~720 µL/L)

Autoclave with stir bar in flask or bottle

Stir until cool enough to handle

Pour 30 mL per plate (use the deep ones)

[1]: Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26

[2]: Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26

Primers

Fisher Custom Oligos

  • Primers come lyophilized.

  • Tube label says how many nmol in the tube (usually ~100-500)

  • Multiply nmol x 10 = μL of sterile water to add to make 100 μM stock.

  • This is the only stock!

  • Put it away safely on instructor shelf in freezer.

  • Make a working stock for students (give them all of it):

  • 12.5 μM (12.5 μL of the 100 μM stock + 87.5 μL water).

(If using the repeater pipet for the water, 85 µL water + 12.14 µL 100 µM stock)

Sea Water

Instant Ocean

  • 34 g/L
  • pulverize in mortar and pestle
  • add very slowly to the water, stirring
  • autoclaving helps the salts go into solution a little

Artificial Seawater, according to Wikipedia

salt molarity
NaCl 0.409
Na2SO4 0.003
KCl 0.009
NaHCO3 0.0023
KBr 0.00082
H3BO3 0.00042
NaFl 0.00007
MgCl2 0.5327
CaCl2 0.01033
SrCl2 0.00009

Stains

Antibodies

DiOC6(3)

Invitrogen 273

DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye that is selective for the **mitochondria:: of live cells, when used at low concentrations. At higher concentrations, the dye may be used to stain other internal membranes, such as the endoplasmic reticulum.

ER Tracker

ER Tracker Red

(BODIPY™ TR Glibenclamide), for live-cell imaging Invitrogen/Life/ ThermoFisher: E34250

Ex = 587 nm Em = 615 nm

MW = 915.23

100 ug lyophilized.

To make stock solution:

  • Add 110 uL DMSO to make a 1mM solution
  • Make 1 uL aliquots

To use

Working concentration: ~1 uM.

  • Add 1 mL HBSS to 1 uL aliquot to make 1uM solution. (Use buffered saline with Ca++ and Mg++)

  • treat cells with warm 1 uM solution 15 - 30 min

  • rinse staining solution out

  • put non CO2 medium in dish

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Fluo-4 AM

Fluo-4 stock
Invitrogen F14217 500 µL

Calcium indicator (fluoresces when bound to Calcium ions)

Ex 494 nm; Em 516 nm

1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution

Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS

Incubate 15 - 60 min at 20 - 37C. Wash before viewing.

20 % w/v Pluoronic

plus HBS to final volume

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Mitotracker

Invitrogen M7512

https://www.thermofisher.com/order/catalog/product/M7513?SID=srch-srp-M7513

Ex = 579 nm; Em = 599

50 ug per tube.

MW = 531

Add 100 uL DMSO to 50 ug in tube. Makes ~ 1 mM stock solution.

Use at 25 - 100 nM. (1 uL per 10 mL for 100 nM)

Dilute in medium. Treat with pre-warmed stain solution for 15 - 45 minutes. Replace staining solution with fresh (warm) medium.

To fix: use 3.7% formaldehyde (in medium); 37C for 15 minutes (??)

NBD Ceramide

Invitrogen N22651

fluorescent marker for Golgi in live cells

Ex: 466nm Em: 536nm

Follow instructions attached: Add 150 µL sterile H2O to the 5 mg in the bottle. (Makes 0.5mM (=500 µM) in BSA)

Aliquot 10 µL, and freeze

Working concentration is 5 µM

Add 990 µL HBS to 10 µL in tube

OR 99µL 10x HBS + 891 µL water

Aliquot ~120 µL per group (makes 8 aliquots)

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PS-Speck

Fluorescent Beads

Molecular Probes P7220

Code Color Ex (nm) Em (nm) filter cube label
A blue 360 440 UV
B green 505 515 B
C orange 540 560 LP (?) G?
D deep red 633 660 probably not
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Phalloidin

General protocol:

Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)

Aliquots

  • 5 µL stock per 0.5 mL tube
  • Label: Add 200 µL PBS (or PBS 1% BSA), use 50µL per coverslip

Fix cells with formaldehyde

Procedure:

  • 50 uL phalloidin on parafilm in humid chamber
  • coverslip cell side down onto drop
  • 15 - 20 minutes at room temp
  • rinse repeatedly in PBS-Tween-azide
  • blot the corner on a kimwipe
  • Mount on a drop of mounting medium on a slide
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FITC phalloidin

Thermo Fisher F432

From the manufacturer:

Fluorescein phalloidin is a high-affinity F-actin probe conjugated to the green fluorescent dye, fluorescein (FITC).

  • Selectively stains F-actin
  • Excitation/Emission: 496/516 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

10 and 20 uL aliquots

In histology freezer

Red phalloidins

Alexa Fluor 568 Phalloidin

Alexa Fluor 568 Phalloidin

Fisher or Invitrogen A 12380

Binds to F-Actin

Ex/Em 578/600

Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)

Aliquots

  • 5 µL stock per 0.5 mL tube
  • Label: Add 200 µL PBS (or PBS 1% BSA), use 50µL per coverslip

Fix cells with formaldehyde

Procedure:

  • 50 uL phalloidin on parafilm in humid chamber
  • coverslip cell side down onto drop
  • 15 - 20 minutes at room temp
  • rinse repeatedly in PBS-Tween-azide
  • blot the corner on a kimwipe
  • Mount on a drop of mounting medium on a slide

In histology and bioimaging freezers

Rhodamine Phalloidin

Thermo Fisher R415

From the manufacturer:

Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC).

  • Selectively stains F-actin
  • Excitiation/Emission: 540/565 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples
  • Very widely cited fluorescent phalloidin conjugate

In 262A freezer

Texas Red-X Phalloidin

Thermo Fisher T7471

From the manufacturer:

Texas Red®-X phalloidin is a high-affinity F-actin probe conjugated to our bright, photostable, red fluorescent Texas Red®-X dye.

  • Selectively stains F-actin
  • Outstanding fluorescence performance
  • Excitation/Emission: 591/608 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

In histology freezer

Syto RNASelect

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

  • Cells on coverslip
  • Warm 500 nM labeling solution to 37C
  • Incubate at 37C 20 min
  • Rinse twice in PBS or medium
  • Add warm medium, let cells rest 5 min
  • Fix in chilled methanol 10 min at -20C
  • Several washes in PBS

Fixed cells

  • Remove coverslip from medium
  • Fix in chilled methanol 10 min at -20C
  • Remove methanol, let slip sit in PBS 5 min
  • Apply labeling solution 20 min RT
  • Wash 5 min in PBS
  • Mount coverslip
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TMRE

Tetramethrylrhodamine ethyl ester perchlorate

mitochondria-specific Red fluophore

Biochemika (Sigma) 87917

Soluble in DMSO, alcohols.

Stock is 50 mM in DMSO

λex 540 nm; λem 595 nm in DMSO

cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria.

Potential-sensitive probe for measuring membrane potential changes in mitochondria

In freezers in 266A and 362A

Tetramethylrhodamine α-Bungarotoxin

Thermo Fisher T1175

Binds to acetylcholine receptor at neuromuscular junction

From the manufacturer:

Tetramethylrhodamine a-bungarotoxin can be used to visualize this receptor. Labeled a -bungarotoxin conjugates can be used to facilitate identification of nicotinic AChRs and to localize neuromuscular junctions.

Excitation⁄Emission (nm): 554⁄577

In freezer 266A

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Transferrin

Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM).

Final concentration = 1 µM.

Aliquot 2.5 µL.

Add 150 µL Fe-HBS-BSA to make 153 µL 1 µM

Store in freezer

Transferrin is a monomeric serum glycoprotein (~80,000 daltons) that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis.

Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron.

Fluorescent transferrin conjugates can therefore be used with fluorescent LDL to distinguish the lysosomally directed and recycling endosomal pathways.

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Stock solutions

Freezer

antibiotics

antibodies

ABA

(+)-cis,trans-abscisic acid

Plant Media 30631017-1 250 mg $68

soluble in EtOH, MeOH, DMSO 20 - 50 mg/mL

MW = 264.3

Store as powder in freezer.

Make 1 mL of 100mM solution:

0.1mol/1000mL x 164.3g/mol = 0.0264 g

Working concentration ~0.15 mM

In 30 mL MS agar, =

vi = 30mL x 0.15 mM/100 mM = 0.045 mL

ATP

10 mM ATP Stock
(from Maniotis)

60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O

Aliquot and freeze.

Use at ~10 µM

Adenine

Fisher AC147440250 25 gr.

1 mg/mL solution for yeast MV-Ade medium

heat and stir for a couple of hours

Antibiotics

Amphotericin

Anti-Fungal

Stock solution: 10 mg/mL in DMSO (insoluble in ethanol)

Store at 4C short term, -20 long term (in 15 mL tube, wrapped in foil with tape label)

Keep in the dark.

Final concentration in medium = 10 ug/mL

Add 1 mL per L

Freezes in the refrigerator.

Protect from light.

Ampicillin

50 mg/mL stock

(Final concentration in medium = 50 µg/mL)

0.5 g in 10 mL water

filter sterilize

1 mL aliquots (enough for 1 L medium)

0.4 mg/mL for QSB

Make 35 mL:

0.28 mL 50 mg/mL stock

35 mL sterile water

(filter sterilize if any doubt about sterility)

48 0.7 mL aliquots

to make: mL amp solution
at this concentration: mg/mL
add: g dry ampicillin

Carbenicillin

(More stable than ampicillin)

Fisher 50841234b 25 mL Teknova C2130

100 mg/mL stock

effective concentration 50 - 100 µg/mL in medium

Freeze aliquots.

Chloramphenicol

MP 19032

store dry at RT

Cipro

Ciprofloxacin 10 mg/mL stock

(final concentration in medium = 10 µg/mL)

100 mg in 10 mL dilute acid (add HCl drop by drop, mixing in between till it dissolves)

filter sterilize

1 mL aliquots

freeze

0.2 mg/mL for QSB

0.7 mL 10 mg/mL stock

35 mL water

filter sterilize

48 0.7 mL aliquots

To make mL 0.2 mg/mL cipro
you'll need: g cipro

freeze

Doxycycline

Tetracycline category

solubility 50 mg/mL in water

disk has 30 ug

Erythromycin

solubility

50 mg/mL in ethanol

10 mg/mL stock

(10 µg/mL in medium)

0.25 g in 25 mL EtOH

1 mL aliquots

freeze

0.6 mg/mL for QSB

2.1 mL 10 mg/mL stock

33 mL EtOH1

48 0.7 mL aliquots

freeze


  1. Try 20 µL in 330 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. 

Kanamycin

50 mg/mL stock (50 µg/mL in medium)

1.25 g in 25 mL H2O

filter sterilize

1 mL aliquots

freeze

1.2 mg/mL for QSB

0.84 mL 50 mg/mL stock

34.16 mL water

filter sterilize (if any doubt about sterility)

48 0.7 mL aliquots

To make mL 1.2 mg/mL kanamycin
you'll need: g kanamycin

freeze

Kan calcs

if Kanamycin stock is: mg/mL
to make: mL medium
with a final Kan concentration of: mg/mL
Add: mL kanamycin stock

Tetracycline

15 mg/mL stock

0.275 g tetracycline

25 mL EtOH

1 mL aliquots

freeze

1.2 mg/mL for QSB

2.8 mL 15 mg/mL stock

32.2 mL EtOH1

48 0.7 mL aliquots

freeze

To make mL 1.2 mg/mL tetracylcine
you'll need: g tetracycline


  1. Try 28 µL in 322 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. 

Triclosan

Irgasan

Sigma 72779-5g-f

MW 289.54

Boric acid

Boric acid (H3BO3) 0.01 M (=10 mM)

1000x for M&S micronutrients

MW = 61.83

0.031 g/50 mL

MW of Boric acid:
Concentration: M
to make: mL
add: g boric acid

Calcium chloride

CaCl stocks in two concentrations:

MW (dihydrate) = 147

MW:
Concentration: M
to make: mL
add: g CaCl2

Calcium nitrate

Ca(NO3)2.4H2O

FW = 236.1

0.4M stock:

94.4g/L = 0.189g/50mL

MW of calcium nitrate:
Concentration: M
to make: mL
add: g calcium nitrate

Cholesterol

5 mg/mL for C. elegans medium

Fisher AAA1147018 Alfa Aesar 50 g ~$35

in 95% ethanol

Stir for 3 hours

or vortex several minutes

Do not autoclave!

Cobalt Chloride

Cobalt Chloride (CoCl2) 105 µM (=~ 0.1 mM)

10,000X for M&S micronutrients

MW (hexahydrate) = 237.93

0.00125 g/50 mL

MW:
Concentration: M
to make: mL
add: g dry stuff

DTT

Dithiothreitol

154.25 MW

1M = 1.5425g in 10 mL H20

MW:
Concentration: M
to make: mL
add: g DTT

EDTA

EDTA (disodium) 0.5 M pH 8.0

MW = 372.4

18.62 g/100 mL

9.31 g/50 mL

initial pH = ~6. Use NaOH pellets to bring to pH = 8. ~2 g NaOH/100 mL

MW:
Concentration: M
to make: mL
add: g EDTA

EGTA

EGTA 0.5M

100 ml solution

19g EGTA (MW 380g/mol) ddH2O to 90ml adjust pH 7.5/8.0 with solid NaOH (>4g) adjust volume to 100ml

MW:
Concentration: M
to make: mL
start with: mL
add: g EDTA, adjust pH to 7.5 or 8 with NaOH pellets, then bring to final volume

Note: EGTA will not go into solution without NaOH. Once the pH has been raised sufficiently it dissolves quickly. For pH 7.5 the exact amount required is slightly above 4g. Add 3.5-4g immediately, then proceed carefully not to overshoot the desired pH.

from http://www.researchgate.net/post/How_can_I_dissolve_EGTA

Ferric citrate

Ferric citrate (C6H5FeO7) 89.4 mM

MW=244.95

1.095 g/50 mL

0.328 g/15 mL

Keep refrigerated

The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.

Maximum solubility is 1 g/100 mL hot H2O

MW of ferric citrate:
Concentration: M
to make: mL
add: g ferric citrate

Could in the future make a 10 mM batch with 0.1225 g in 50 mL, and for this lab: https://wahoo.nsm.umass.edu/content/reagents-61 , you would have easier calculations.

Ferrous sulfate

Ferrous sulfate (FeSO4) 10 mM

MW (heptahydrate) = 278.01

MS 10x = 28mg/L = 0.1 mM - 100 µM

MS 1x = 0.01 mM = 10 µM

make 1,000x for M&S medium = 0.01 M = 10 mM

MW of FeSO4:
Concentration: M
to make: mL
add: g FeSO4

Keep refrigerated

Turns yellow. Probably oxidation. Yellow comes off on a micropore filter.

Lithium Chloride

LiCl 6M

MW = 42.39

MW of LiCl:
Concentration: M
to make: mL
add: g LiCl

Gets hot!!

MES

MW = 195.2

150 mM stock solution for LPGM

MW of MES:
Concentration: M
to make: mL
add: g MES

Magnesium chloride

Magnesium chloride (MgCl2) 1M

MW (hexahydrate) = 203.31

10.166 g/50 mL

20.33 g/100 mL

MW of MgCl2:
Concentration: M
to make: mL
add: g MgCl2

Magnesium sulfate

Magnesium sulfate (MgSO4) 1M

MW (heptahydrate) = 246.48

MW of MgSO4:
Concentration: M
to make: mL
add: g MgSO4

Manganese chloride

MnCl2 4H2O

M.W.197.9

5 mM

0.0495g/50mL

MW of MnCl2:
Concentration: M
to make: mL
add: g MnCl2

Manganese sulfate

MnSO4

MW (monohydrate) = 169

store dry in dessicator

MS 10X micronutrients: 16.9 mg/L = 0.1 mM

so 1X = 0.01mM = 10 µM

1000X MS = 10 mM

MW of MnSo4:
Concentration: M
to make: mL
add: g MnSO4

Mannitol

MW = 182.172

Concentration: %
to make: mL
add: g mannitol

Potassium acetate

Potassium acetate (C2H3O2K)

Fisher BP364-500 $46.30

MW = 98.14

49 g in 100 mL H2O

MW of KOAc:
Concentration: M
to make: mL
add: g KOAc

Potassium chloride

Potassium chloride (KCl)

MW = 74.56

MW of KCl:
Concentration: M
to make: mL
add: g KCl

Potassium ferricyanide

Potassium ferricyanide (K3Fe(CN)6) 50 mM (= 0.05 M)

red salt

MW = 329.26

0.8232 g/50 mL

MW of Potassium ferricyanide:
Concentration: M
to make: mL
add: g Potassium ferricyanide

Store frozen

Potassium ferrocyanide

Potassium ferrocyanide (K4Fe(CN)6) 50 mM (= 0.05M)

yellow salt

MW (trihydride) = 422.41

MW of Potassium ferrocyanide:
Concentration: M
to make: mL
add: g Potassium ferrocyanide

Store frozen

Potassium hydroxide

KOH

MW = 56.11

MW of KOH:
Concentration: M
to make: mL
add: g KOH

Use for adjusting PIPES pH

Potassium nitrate

Potassium nitrate (KNO3)

MW of KNO3:
Concentration: M
to make: mL
add: g KNO3

Store in refrigerator

Potassium phosphate dibasic

K2HPO4

MW = 174.18

MW of K2HPO4:
Concentration: M
to make: mL
add: g K2HPO4

Potassium phosphate monobasic

Potassium phosphate monobasic (KH2PO4)

MW of KH2PO4:
Concentration: M
to make: mL
add: g KH2PO4


for C. elegans medium:

pH to 6.0 with solid KOH

(~1.7 g/100 mL)

Sterilize (can be autoclaved)

SDS

SDS 20%

Sodium dodecanesulfate (=Sodium lauryl sulfate, NOT laureth)

CH3(CH2)11OSO3Na

Concentration: %
to make: mL
add: g SDS

DO NOT AUTOCLAVE - it (like all detergents) can boil over

Sodium Ferric EDTA

NaFe(III)EDTA

FW = 367.05

50 mM

MW of NaFe(III)EDTA:
Concentration: M
to make: mL
add: g NaFe(III)EDTA

Sodium acetate

Sodium acetate (NaOAc) 3M

CH3COONa

Make new for 2016-2017

MW (trihydrate) = 136.08

MW of NaOAc:
Concentration: M
to make: mL
add: g NaOAc

pH 5.2

Sodium azide

For PBS-Tw-Azide

NaN3

MW = 65

10% w/vol

Concentration: %
to make: mL
add: g NaN3

Sodium bicarbonate

Sodium bicarbonate (CHNaO3) 0.5 M (=500 mM)

MW = 84.01

MW of Sodium bicarbonate:
Concentration: M
to make: mL
add: g sodium bicarbonate

Write the date on it. Probably only stable for 2 weeks.

Sodium chloride

Sodium Chloride (NaCl) 5 M

MW = 58.44

Concentration: M
to make: mL
add: g NaCl

Sodium phosphate dibasic

MW of Na2HPO4:
Concentration: M
to make: mL
add: g dry stuff

Use hot water

Sodium phosphate monobasic

Sodium phosphate monobasic (NaH2PO4)

MW (monohydrate) = 137.99

MW of NaH2PO4:
Concentration: M
to make: mL
add: g dry stuff

Sodium sulfate

Na2SO4.10H2O

MW = 322.2

MW of Na2SO4:
Concentration: M
to make: mL
add: g Na2SO4

Spermidine

Spermidine:

  • neutralizes and stabilizes the negative charge on the DNA phosphate backbone
  • displaces other ions from DNA
  • stimulates restriction enzyme reactions
  • increases specificity and reproducibility of Taq-mediated PCR

Used in Gene & Genome

Tris

Tris 1M

Tris base MW = 121.14

MW of Tris:
Concentration: M
to make: mL
add: g Tris

pH to 7.5 or 8.0, depending on the application

Triton X

Triton X 10%

MW = 646.86

1 g = ~1 mL

Concentration: %
to make: mL
start with: mL Triton X

Tween 10%

Tween 10%

for PBS-Tween-Azide

1 g = ~1 mL [density = 1.095 g/mL at 25 °C (from Sigma website)]

Concentration: %
to make: mL
start with: mL Tween

Do not autoclave

Sugars

Glucose

MW = 198.17

MW of Sucrose:
Concentration: M
to make: mL
add: g sucrose

Lactose

MW = 342.3

MW of Sucrose:
Concentration: M
to make: mL
add: g sucrose

Mannitol

MW = 182.17

MW of Sucrose:
Concentration: M
to make: mL
add: g sucrose

Sucrose

MW = 342.3

MW of Sucrose:
Concentration: M
to make: mL
add: g sucrose

Yeast media

MV + Ade

  • 0.15 g YNB
  • 0.52 g ammonium sulfate
  • 2.0 g agar
  • 82 mL H2O
  • 8.0 mL 1 mg/mL adenine
  • 10.0 mL 20% glucose (final conc = 2g/100 mL)

Minimal Vitamin Medium plus Adenine

Label plates with "+ ADE"

To make MV+ADE agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g YNB*, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
add asceptically: mL filter-sterilized Adenine (1mg/mL)
for a final volume of mL

*Yeast Nitrogen Base without amino acids and ammonium sulfate

MV

Minimal Vitamin Medium

To make MV agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g Yeast Nitrogen Base without amino acids and ammonium sulfate, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

Mark with a single blue line

YED

Yeast Extract Dextrose

To make YED agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

Mark with a single red line.

YEKAC

Sporulation Medium

To make YEKAC agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g potassium acetate, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes

Mark with a single black line.

YPD

Yeast extract-Peptone-Dextrose medium:

To make YPD agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone broth, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL YPD agar