Table of Contents








1G4F Fixative
72 mL tapwater
100 mL filtered seawater
24 mL 37% stock formaldehyde
4 mL 50% gluteraldehyde

Acid Alcohol
Mix 0.2% HCl into 70% EtOH

AFA Fixative
10 p formalin
50p 95% EtOH
2p acetic acid
40p Se2H2O

Ammonium Hydroxide
Make fresh daily
1-2 mL concentrated ammonium hydroxide
250 mL tap water

Ampicillin Stock Solution
200X (20mg/mL)
  1. Dissolve 20 mg/ml (ie. 1g in 50 ml) ampicillin in dH2O.
  2. Use syringe to filter sterilize it.
  3. Store in sterile conical tube in refrigerator.
  4. Add to autoclaved media at a 1:200 dilution, after media cools down to 50°C.
  5. Throw out stock solution and any media with ampicillin after 4 weeks.


Use the chart below for some commonly used volumes.

200X stock
Media: agar or broth
Final Concentration
5 ml
1 liter
100 ug/ml
0.5 ml
100 ml
100 ug/ml
50 ul
10 ml
100 ug/ml

Anitbody Elution Buffer
Gentle, removes 2° Ab, 3 X 15 mins
5 mM Glycine: 0.188 g
500 mM NaCl: 14.61 g
0.5% Tween 20: 2.5 mL
100 ug BSG/mL: 50 mg
In 500 mL, pH 2.3 without 1 N HCl

Harsh, removes 1° and 2° Ab
2% SDS: 0.5g
100mM B-mercapto-etoh: 1.76 uL
63 mM Tris base, pH 6.8: 0.191 g
In 25 mL (enough for 1 block)

  1. Wash blot 2X, 5 min PBST
  2. Incubate 30 min, 50° in elution buffer
  3. Wash 3X, 5 min PBST
  4. May need to repeat

Antibody Solution
For In-situ

For 7 mL solution
Alkaline Phosphate (AP)-labeled sheep
anti-DIG antibody conjugate diluted 1:1000
0.007 mL
Buffer 1
6.909 mL
1% sheep serum
0.07 mL
0.3% Triton X-100
0.021 mL

APS
10%, make fresh
.02 g APS
200 mL uL nH2O

Bacto-Agar Soup Recipe
2ml F/2
2ml Silicate solution (3g sodium metasilicate pentahydrate in 1L DI water)
10ml Tris solution (100g trisma base, 930ml of DI water, 70ml muriatic acid)
1L filtered (0.22µm) seawater
2g Bacto-Agar powder

Stir ingredients in large flask over heat until boiling. Pour 200ml of the media into 5 flasks and autoclave. Allow media to cool overnight. Once cooled, add 2ml of inoculant to each of the 5 flasks of media (1:100 ratio). Cover flasks with saran wrap and label with contents, date, and name. Store cultures in 15°C incubator with constant light. Every month 2ml of the “old soup” media should be mixed with 200ml of fresh media to keep the nitzchia happy.

Blocking Buffer
For In-situ, 500 uL blocking buffer/slide

For 10 mL solution
2% Sheep serum
0.2 mL
0.3% Triton X-100
0.03 mL
Buffer 1
9.77 mL

Blotto Blocking Buffer
To block nitrocel. membrane
9g nonfat milk in 100 mL TBST

Bouin's
750 mL picric acid, saturated aqueous solution (71.4%)
250 mL 37% formaldehyde (23.8%)
50 mL glacial acetic acid (4.8%)

Bouin's Fixative
1 L, Made fresh daily
Dissolve 2.0 g picric acid into 500 mL dH2O
Filter through Whatman No. 1
Add 20g paraformaldhyde
Heat to 60 deg C
Add several drops of NaOH to help dissolve
Cool
Add 500 mL 2x PBS
Add 0.48g KH2PO4
pH to 7.2

Bromophenol Blue
.02 g
100 mL nH2O

Buffer 1
For In-situ, pH 7.5

For 1000mL solution
100 mM Tris-HCl
15.76 g
10 mM NaCl
0.5884 g

Buffer 2
For In-situ, pH 9.5

For 1000 mL solution
100 mM tris-HCl
15.76 g
100 mM NaCl
5.844 g
50 mM MgCl2
4.76 g

Buffered Neutral Formalin
10%
100 mL full strength formalin
6.5 g sodium phosphate dibasic anhydrous
4.0 g sodium phosphate monobasic
900 mL dH2O

Cloney's Working Solution
Make fresh daily
5 mL 25% gluteraldehyde
25 mL 0.4M phosphate buffer
20 mL 0.34M sodium chloride

Colloidal Blue-G
0.1% Coomassie (Serva) Blue G in 12.5% TCA
500mg Blue G in 78 mL 80% TCS (stock) + 422 mL H2O

No need to de-stain! Only binds protein doesn’t stain gel. Should be “army green”, if it goes blue add more acid.

Counter stain
For In-situ, 0.05% Bismarck Brown Y
Weigh 0.05 g of Bismarck Brown Y.
Mix in Nano H2O 100 ml

Dolly suggested to dilute Bismarck Brown Y 0.05% to 1:10 in PBS (Neutral pH)

Davidson's Invertebrate Fixative
95% Ethanol
30%
1200 ml
Formaldehyde
20%
800 ml
Acetic Acid
10%
400 ml
dH2O
18%
720 ml
Seawater
12%
480 ml
Glycerol
10%
400 ml

Davidson's Shrimp Fixative
95% Ethanol
330 ml
Formaldehyde
220 ml
Glacial Acetic Acid
115 ml
Tap Water
335 ml

Davidson's Vertebrate Fixative
1.8 L
95% Ethanol
600 ml
Formaldehyde
400 ml
Glacial Acetic Acid
200 ml
dH2O
600 ml
*If you won't be fixing often you can omit the glacial acetic acid and add 10% glacial acetic acid just prior to use

Dextran Sulfate
50x, 10 mL
Weigh 5 g in NanoH2O
Then stir for 3 min.

Drying Solution
20% EtOH
10% Glycerol
70% npH2O to 1L

EDTA
0.5M, pH 8.0
186.1 EDTA
18-20 g NaOH
1-4 mL 10M NaOH to pH
Fill to 1 L with dH2O

Ethanol

For 250 mL
95%
237.5 mL 100% EtOH + 12.5 mL H2O
80%
200 mL 100% EtOH + 50 mL H2O
70%
175 mL 100% EtOH + 75 mL H2O
50%
125 mL 100% EtOH + 125 mL H2O

Ficoll based loading buffer
15% Ficoll (type 400) polymer in distilled water
0.25% Bromophenol Blue
0.25% Xylene cyanol FF

Storage: Room Temperature

Formaldhyde Buffered
37%For 1G4F, stock solution, versitile for light and Em microscopy
90 mL formaldhyde
1.38 g Na2HPO4
.0056 g NaOH
Store at 4 deg C

Formaldhyde
For Hawkes' fixative
Dissolve 12g paraformaldehyde flakes in 125 mL dH2O at 60-70 deg C, adding NaOH solution (1 pellet dissolved in 5 mL dH2O) dropwise until paraformaldehyde is dissolved

Formaldhyde/PBS
3.5%, made fresh daily
Add 7g paraformaldehyde to 100mL dH2O
Heat to 60 deg C
Add a few drops of NaOH to hep dissolve
Filter through Whatcom No. 1
Cool to 20 deg C
Add 100mL 2x PBS
Store at 4 deg C

G-250
500 mg Serva-based Coomassie Blue G
200 mL MeOH
250 mL HAc
50 mL H2O

De-stain: 10% MeOH + 10% HAc
Store: 7% HAc

Gel De-stain
300 mL HAc (10%)
300 mL MeOH (10%)
To 3 L npH2O

Hawkes' Buffer
Sodium cacodylate, 0.1M in dH2O
pH to 7.4 with 1N HCl
Sucrose, 5.5%
CaCl2, 0.02%
Store in sterile bottle at 4 deg C

Hawkes' Working Solution
30 mL gluteraldehyde 25%
120 mL formaldehyde solution
900 mL Hawkes' buffer

Hematoxylin
Filter Harris' Alum Hematoxyling through a Whatcom No. 1 filter prior to use

Karnovski's Fixative
1/2 strength, for light and electron microscopy
40 mL 37% formaldehyde
40 mL 25% glutaraldehyde
100 mg picric acid
2.722 KH2PO4 (MW=136.09 g)
11.36 g Na2HPO4 (MW=141.96 g)
Add NPH2O to 1000 mL

KCL Buffer
Final
g/mol
g/500mL
g/250mL
g/100mL
5mMMgSO4
247.48
0.616
0.308
0.1232
5mM NaH2PO4*2H2O(dihydrate)
155.99
0.390
0.195
0.0780
40mM HEPES
238.3
4.766
2.383
0.9532
150mM sorbitol
182.17
13.663
6.832
2.7326
Adjust pH to 7.55 @ 37 deg C with either 70mM KCL (0.522 g/1oo mL + 300 uL 10 N KOH/100 mL) OR
40mM KCL if you want fnial [K+} = 70 mM and [CL-] = 40 mM (0.298 g/100 mL + 300 uL 10 N KOH/100 mL)

Store in sterile 15mL aliquots at 4 deg C

KGB Buffer
365 mOsm/kg
Final
g/mol
g/500mL
g/250mL
g/100mL
5mMMgSO4
247.48
0.616
0.308
0.1232
5mM NaH2PO4*2H2O(dihydrate)
155.99
0.390
0.195
0.0780
40mM HEPES
238.3
4.766
2.383
0.9532
70mM K+ gluconate (under gluconic acid)
234.2
8.197
4.098
1.6394
150mM sorbitol
182.17
13.663
6.832
2.7326
Starting pH will be ~6.7, adjust pH to 7.55 @ 37 deg C with 10N KOH (~300 uL/100 mL adds 30mM K+)
Adjust pH to 7.4 for Artemia
Store in sterile 15mL aliquots at 4 deg C

KH2PO4 Buffer
0.1M
13.6g KH2PO4 powder
1L NP H2O
pH to 4.5
Flter sterilize

Laby broth
For Labyrinthula zostrae media broth
1. We use per 1 L 0.45um filtered seawater, adjusted to 25ppt:
  • Germanium dioxide: 1.5 mg
  • Yeast extract: 0.1g
  • Peptone: 0.1g
  • Glucose: 1.0g
2. Split into four 250 mL corning jars (the ones with the orange lids)
3. Autoclave for 15-20 minutes.
4. So after autoclaving, Temper the media for 30 minutes to between 55 and 60 C with stirring. Using sterile technique then add the following to each jar:
  • Horse Serum: 2.5 mL
  • Pen/Strep: 6.25 mL
5. Store at 4 deg C in clean room fridge until use

Laby plates
For Labyrinthula zostrae culture plates
1. We use per 1 L 0.45um filtered seawater, adjusted to 25ppt:
  • "USB Nobel Agar": 12g
  • Germanium dioxide: 1.5mg
  • Yeast extract: 0.1g
  • Peptone: 0.1g
  • Glucose: 1.0g
2. Autoclave for 15-20 minutes. It is helpful to place an autoclaveable magnetic stir bar in the flask before autoclaving so you can stir the medium after autoclaving when you add the pen/strep and horse serum.
3. So after autoclaving, Temper the media for 30 minutes to between 55 and 60 C with stirring or if you have a water bath mixer just gently agitate to avoid creating bubbles in the agar. Using sterile technique then add:
  • Horse Serum: 10mL
  • Pen/Strep: 25mL
4. Stir adequately and pour the plates.


Low TE Buffer
200 mL, for diluting DNA
40 uL 0.5M EDTA
2 mL 1 M Tris
Fill to 200 mL nH2O
Note: 0.5M EDTA not soluble until pH reaches 8.0. Use NaOH to reach pH 8.0.

MEM 5 T
50 ml
100 ml
200 ml
500 ml

AUTO-POW
45.8
91.6
183.2
458

FBS
2.5
5
10
25

L-glutamine
0.5
1
2
5

Tris Buffer
0.7
1.4
2.8
7

Penn-Strep (ml)
0.5
1
2
5

Gentamycin Sulfate (ml)
0.16
0.32
0.64
1.6

Fungizone (ml)
0.4
0.8
1.6
4

MEM 10 SB
50 ml
100 ml
200 ml
500 ml

AUTO-POW
45.8
91.6
183.2
458
FBS
5.2
10.4
20.8
52
L-glutamine
0.5
1
2
5
Sodium Bicarb Buffer
0.6
1.2
2.4
6
Penn-Strep (ml)
0.5
1
2
5
Gentamycin Sulfate (ml)
0.16
0.32
0.64
1.6
Fungizone (ml)
0.4
0.8
1.6
4

Millonig's phosphate buffer stcok
For Cloney's fixative, 0.4 M
11.08 g Sodium phosphate, monobasic (NaH2PO4H2O)
2.85 g Sodium hydroxide
200 mL dH2O
pH to 7.5

Modified TAE
0.1 mM Na2EDTA (working solution)

Neutral Buffered Formalin
10%
Use a commercial 10%, phosphate-buffered (pH 7.0) formaldehyde solution

PBS
(10X)
NaCl 40 g
KCl 1 g
Na2HPO4 5.25 g
KH2PO4 1 g
Add dH2O to final volume of 500 ml to get 10X PBS.
Add 4.5 L dH2O to yield 5 L of 1X PBS.
Adjust pH of 1X PBS to 8.0

PBS
For Avidin/Biotin Alkaline Phosphatase (ABC-AP) detection
10mM dibasic sodium phosphate
0.9% NaCl
pH to 7.5 with phosphoric acid

PBST
(0.05% w/v)
Add 0.5 ml of Tween 20 per 1 L of PBS.

PBST + 1% BSA
Add 0.05 g BSA to 5 ml of PBST

PBST + 2% BSA
Add 0.1 g BSA to 5 ml of PBST

Phosphate-buffered Saline
1 L
Dissolve following into 800 mL dH2O:
  • 8.0 g NaCl
  • 0.2 g KCl
  • 1.44 g Na2HPO4
  • 0.24 KH2PO4
pH to 7.2
Autoclave

Picro-eosin
4.0 g eosin
360 mL 80% EtOH
After dissolved, add 40 mL saturated aqueous picric acid
Filter through a Whatcom No. 1

Prehybridization Buffer
For In-situ

For 50 mL
0.51 mL deionized formamide
25.5 mL
0.2 mL 20x SSC
10 mL
0.05 mL heat-denatured sperm DNA (at 10 mg/mL)
2.5 mL
0.2 mL 50% dextran sulfate
10 mL
0.02 mL 50x Denhardt’s solution*
1 mL
Add Poly A at 0.1 mg/mL

* In order to make denhardt’s solution, take the full bottle of 150 mg of powder and reconstitute with 5 mL of water.

R-250
500 mg Serva-based Coomassie Blue G
400 mL MeOH
80 mL HAc

Fast De-stain: 40% MeOH (EtOH) + 10% HAc
Slow De-stain: 10% MeOH (EtOH) + 10% HAc
Store: 7% HAc

Resolving Gel
12% resolving gel from 30% acrylamide/bis stock (2 mini gels)


0.75mm
1.0mm
1.5mm
ddH2O
3.35 mL
4.02 mL
5.352 mL
1.5M Tris-HCL, pH 8.8
2.5 mL
3.0 mL
4.0 mL
Acrylamide/Bis
4.0 mL
4.8 mL
6.4 mL

degas
degas
degas
10% SDS
100 uL
120 uL
160 uL
10% APS (fresh)
50 uL
60 uL
80 uL
TEMED
5 uL
6 uL
8 uL

Running Buffer
5x, for SDS electrophoresis

Tris Base (25mM) 15.0 g
Glycine (192 mM) 72.0 g
SDS 5.0 g
To 1 L with npH2O
Running buffer should be ~8.3. Check but do not adjust with acid or base.

Dilute to 1X: 60 mLs 5X + 240 mL npH2O


Sample Bufer
2x, for electrophoresis, store in fridge

Stock Solution w/o B-mercaptoethanol:
2.5 mL 0.5M Tris-HCL, pH 6.8
4.0 mL 10% (w/v) SDS
0.25 mL 0.2% bromophenol blue in nH2O
2.0 mL glycerol
0.25 mL nH2O


Add 0.1 mL B-mercaptoethanol to 0.9 mL stock solution for denaturing 2x SB
Add 1:1 to sample, boil 5 min, load onto gel.

Seawater Formalin Fixative
10%, 1L
270 mL 37% formaldehyde
Add seawater to 1000 mL

Seawater Glutaraldehyde Fixative
2.5%, 1L
50 mL 50% glutaraldehyde
Add seawater to 1000 mL

Seawater Nutrient
1ml F/2
1ml Silicate solution
1L filtered (0.22µm) sterile seawater (autoclave seawater prior to the addition of nutrients)

Sodium Bicarbonate Buffer
(7.5% w/v)

50mL
500 mL
npH2O(mL)
50
500
Sodium Bicarbonate(g)
3.75
37.5

Sodium Cacodylate Solution
For Torrence's Fixative, 0.8 M
17.12 g (CH3)2AsO2Na H2O in dH2O to 100 mL

Sodium Chloride Solution
For Cloney's Fixative, 0.34 M
3.97 g sodium chloride in 200 mL dH2O

SSC
For In-situ

1x, pH 7.0

For 1000mL sol
0.15 M NaCl
8.765 g
0.015 M sodium citrate
4.4115 g

2x, pH 7.2

For 1000mL sol
0.3 M NaCl
17.53 g
0.03 M sodium citrate
8.823 g

20x, pH 7.2

For 1000 mL solution
3M NaCl
175.32 g
0.3M Na citrate
88.23 g

Stacking Gel
6.2%, enough for 2 gels with any 0.75 mm or 1.0 mm comb with 1.5 prep-well comb

.75 mm or 1.0 mm combs
.75 mm or 1.0 mm combs
1.5 mm combs

40%
30%
30%
ddH2O
2.316 mL
2.116 mL
3.174 mL
0.5M Tris-HCL, pH 6.8
1.0 mL
1.0 mL
1.5 mL
Acrylamide/Bis
0.6 mL
0.8 mL
1.2 mL

degas
degas
degas
10% SDS
40 uL
40 uL
60 uL
10% APS (fresh)
40 uL
40 uL
60 uL
TEMED
4 uL
4 uL
6 uL

Substrate
45 ul NBT (nitroblue trazolium)
35 ul BCIP
10 mL of Buffer2

Substrate Buffer
For Avidin/Biotin Alkaline Phosphatase (ABC-AP) detection
100mM Tris-HCl
pH to 8.2, in dH2O

Substrate Mixture
For Indirect Immunoperoxidase Detection, use immediately
0.1 mL 3% H2O2
10 mL 50mM containing 6.0 mg of 3,3' Diaminobenidine tetrahydrochloride

Sucrose Solution
For Torrence's Fixative, 0.55 M
18.82 g sucrose in dH2O to 100 mL

TAE
(Tris/acetate/EDTA) – 50X stock solution
242 g Tris base
57.1 ml glacial acetic acid
37.2 g Na2EDTA·2H2O
npH2O to 1 liter

TBE
(Tris/borate/EDTA) – 10X stock solution
108 g Tris base
55 g boric acid
40 ml 0.5M EDTA (pH 8.0)
npH2O to 1 liter
Store stock solutions in a sterile container.
Stir solution for 15-30 minutes.
Note: After solution has been stored for some time, a precipitate may form. If so, discard and make new stock.

TBS
(Tris-buffered saline) -- 1L 50mM
Dissolve 8.0 NaCl, 0.2 g KCl, and 6.0 g Tris base in 800 mL dH2O
pH to 7.6
Autoclave

TBST
(Tris-buffered saline + Tween 20) -- 1L
Dissolve 8.766 g/L of 150 mM NaCl and 1.2114 g/L of 10 mM Tris base
Fill to 1 L with npH2O
pH to 7.4 with 10N HCl (~16 drops/L)
1.0 mL/L 0.1% Tween 20

Torrence's Working Solution
10 mL 8% gluteraldehyde
10 mL 0.8M sodium cacodylate solution
20 mL 0.55M sucrose solution
0.4 g tannic acid (added just before use)
pH to 7.4 just before use

Transfer Buffer
For Western Immunoblotting

1 L
1.5 L
2 L
25 mM Tris Base
3.03 g
4.545 g
6.05 g
192 mM Glycine
14.4 g
21.6 g
28.8 g
17.5% MeOH*
175 mL
262.5 mL
350 mL
Do not adjust pH but check to see that it is >8.0 (8.1-8.4) before adding MeOH
*May need to adjust MeOH depending on molecular weight of protiens.

Tris Buffer
1 M

50 mL
500 mL
1000 mL
npH2O(mL)
50
500
1000
TRIZMA base (g)
2.3
26
46
TRIZMA HCl (g)
4.88
48.8
97.6

Tris Buffer
For In-situ, pH 7.2

For 1000ml sol
0.2 M Tris-HCl
31.52 g
2 mM CaCl
1.5106 g

Tris-HCl
For resolving gel, 250 mL

For .5 M, pH 6.8
For 1.5 M, pH 8.8
Tris
15.14 g
45.41 g
nH2O
bring to 250 mL
bring to 250 mL
pH to:
6.8
8.8

TSB Media
.15 g TSB powder
5mL NP H2O

Yeast Autolysate
  1. 1. Suspend one pound (450 grams) in 500 ml of water.
  2. 2. Incubate at 50C for 24 hours with occasion or mechanical stirring to autolyse the cells.
  3. 3. Let stand in the cold, and decant and use the supernautant fluid.
  4. 4. Use 2 to 10 ml per liter of finished medium to supply growth factors.
  5. 5. Use larger volumes to supply Nitrogen and Carbon sources.

Yeast Infusion Broth
  1. 1. Suspend one pound of fresh baker's yeast in two liters of water.
  2. 2. Autoclave for one hour in a large container.
  3. 3. Allow to settle in the cold and decant the clear supernautant fluid
  4. 4. If desired, clarify with egg white and cooking and filtering thru paper.
  5. 5. Add 1% glucose, if desired. Add 1% agar if desired. add 1% calcium carbonate if desired.

Zenkers Solution
1000 mL dH2O
50 g mercuric chloride
25 g potassium dichromate
10 g sodium sulfate