Recipes

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.

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:

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 49.1 50.3
7.0 61.5 38.3
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

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.

Bacterial media

LB agar

25 mL/plate

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

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

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

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

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

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

Plant growth media

MS high salt

150 mM NaCl

Make regular MS, then add 0.03 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

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)

Stock solutions

Freezer

antibiotics

antibodies

Inventory

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                  <div class="breadcrumb"><a href="/biology/">Home</a> › <a href="/biology/faculty">Faculty</a> › Current Faculty</div>                                      <h1 class="title">Current Faculty</h1>
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  <p>Below is an alphabetical listing of Current Biology faculty. Please note you can search by first or last name. All of Biology faculty are affiliated with at least one of the four <a href="/biology/graduate/interdisciplinary-graduate-programs">interdisciplinary graduate programs</a> and you can limit your search to only those faculty affiliated with a particular program.  In addition, you can search for only those faculty who can chair graduate student Masters or PhD committees.  And you can combine these criteria to further limit your search.</p>
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Lynn Adler

Associate Professor

Ecology and evolution of insect-plant interactions

R. Craig Albertson

Assistant Professor

Evolutionary Developmental Biology

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

Antibiotics

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 0.4 mg/mL ampicillin
you'll need: g ampicillin

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

Erythromycin

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

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

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

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 = 10,000X for M&S medium

Keep refrigerated

Lithium Chloride

LiCl 6M

MW = 42.39

10 mL: 2.543 g

15 mL: 3.815 g

25 mL: 6.3585

50 mL: 12.717

Gets hot!!

Magnesium chloride

Magnesium chloride (MgCl2) 1M

MW (hexahydrate) = 203.31

10.166 g/50 mL

20.33 g/100 mL

Magnesium sulfate

Magnesium sulfate (MgSO4) 1M

MW (heptahydrate) = 246.48

12.324 g/50 mL

19.718 g/80 mL

Manganese chloride

MnCl2 4H2O M.W.167.27

1 M 8.364 g/50 mL

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

Mannitol

MW = 182.172

Concentration: %
to make: mL
add: g mannitol

Potassium acetate

Potassium acetate (C2H3O2K) 5M

Fisher BP364-500 $46.30

MW = 98.14

49 g in 100 mL H2O

Potassium chloride

Potassium chloride (KCl) 1M

MW = 74.56

3.728 g/50 mL

7.456 g/100 mL

Potassium ferricyanide

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

red salt

MW = 329.26

0.8232 g/50 mL

Store frozen

Potassium ferrocyanide

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

yellow salt

MW (trihydride) = 422.41

1.056 g/50 mL

store frozen

Potassium hydroxide

KOH

MW = 56.11

5M solution:

14.0275 g/50 mL

Use for adjusting PIPES pH

Potassium nitrate

Potassium nitrate (KNO3) 1M

Store in refrigerator

Potassium phosphate dibasic

K2HPO4

1M

MW = 174.18

8.709 g/50 mL

17.418 g/100 mL

To make mL 1M K2HPO4
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

10 g in 50 mL

20 g in 100 mL

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

Sodium acetate

Sodium acetate (NaOAc) 3M

CH3COONa

MW (trihydrate) = 136.08

2.04 g/15 mL

6.08 g/50 mL

pH 5.2

Sodium azide

For PBS-Tw-Azide

NaN3

MW = 65

10% w/vol

2.5 g in 25 mL water

Sodium bicarbonate

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

MW = 84.01

4.2 g/50 mL

8.4 g/100 mL

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

Sodium chloride

Sodium Chloride (NaCl) 5 M

MW = 58.44

14.61 g/50 mL

29.22 g/100 mL

146.1 g/500 mL

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) 1M

MW (monohydrate) = 137.99

6.9 g/50 mL

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

sodium phosphate

Sodium phosphate buffer pH 7.2 1M

Sodium sulfate

Na2SO4.10H2O 1M

MW = 322.2

16.11 g in 50 mL

32.22 g in 100 mL

Spermidine

Tris

Tris 1M

Tris base MW = 121.14

12.114 g/ 100 mL

pH to 7.5 or 8.0, depending on the application

Triton X

Triton X 10%

MW = 646.86

5 g (=~ 5 mL) + 45 mL H2O

Tween 10%

Tween 10%

for PBS-Tween-Azide

density = 1.095 g/mL at 25 °C (from Sigma website)

10 mL Tween/ 100 mL water (or PBS)

Do not autoclave

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

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

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.