RECIPES FOR LABORATORY SOLUTIONS

30% Acrylamide Solution (0.8% bis)
60 g acrylamide
1.6 g bis
Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle.
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50% Acrylamide Solution (19:1 acrylamide:bis)
237.5 g (97%) acrylamide
12.5 g bis
Dissolve in 250 ml hot water. Filter to remove debris and wrap in foil.
Alkaline Phosphatase Buffer
100 mM Tris-HCl, pH 9.5
100 mM NaCl
5 mM MgCl2
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 5 7.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLAVE!
Ampicillin Stock Solution, 100mg/ml
1.00 g sodium ampicillin (stored in 4° dessicator; Bristol Polycillin-N, 1g/vial; or Sigma
10 ml of autoclaved water
Store at - 30°C in sterile Falcon tube
20% Bac- Acrylamide Solution     WARNING: wear gloves
100 ml solution
18.98 g acrylamide
1.02 g BAC (N',N'-bis-acrylylcystamine)
Heat approximately 75 ml of water to almost boiling. Add acrylamide and cover with a watch glass. Mix briefly to dissolve and reheat to near boiling. Add BAC. QS to 100 ml with water. Do not autoclave. Filter sterlize with Nalgene 0.45 or 0.2 micron filter and store in foil wrapped bottle.
Bac teriophage Dialysis Buffer
0.1 M Tris-HCl, pH 7.5 (50 mls of 1M stock)
0.5 M NaCl (50 ml of 5M stock)
2.5 mM EDTA (2.5 ml of a 0.5M stock)
QS to 500 ml with water and autoclave.
Base Treatment Solution (1.5M NaCl, 0.5M NaOH) for Southern and Benton Davis blots.
For 2 liters:
175.35 g NaCl
40 g NaOH
QS to 2 liters with water and autoclave.
5X Borate Buffer (0.9 M Tris, 0.9 M Boric Acid, 0.025 M EDTA, pH 8.3)
For 2 Liters:
218.0 g Tris
111.29 g H3B03
18.61 g EDTA
pH to 8.3; QS to 2 liters and autoclave. (See notes on 10x TBE recipe regarding precipitation on storage).
Balance Salt Solution (BSS)
Stock Solution 1
3.0 g dextrose
0.6 g KH2PO 2(H2O)7
1.07 g Na2HPO4(H2O)7
6 ml 0.5% phenol red solution
Dissolve and bring to 300 ml with double distilled water.
Stock Solution 2
558 mg CaCl2(H2O)2
1.2 g KCl
24 g NaCl
0.6 g MgCl2(H2O)6
0.6 g MgSO4(H2O)7
Dissolve and bring to 300 ml with double distilled water. Let the stock solutions stand overnight at 4°C. They may be stored at 4°C or at room temperature. The pH of stock solutions should be around 7.2.
To prepare 3 L of BSS, add 300 ml of stock solution 1 to 2.4 L of water and mix. Add 300 ml of stock solution 2 to the dilute stock solution 1 and mix. Filter sterilize. Incubate for 1 week to check for contamination. Store at room temperature.
Cell Lysis Buffer A
0.5% sodium N-lauroyl sarcosinate (helps solubilize proteins; S igma L-5125)
10 mM EDTA
50 mM Tris-HCl, pH 8.0
14C India Ink
Prepare at 10 µCi/ml of ink. We usually use a labelled amino acid or glucose.
50 mM Calcium Chloride
Dissolve 1.84 g CaCl2 in 250 ml H2O. Autoclave.
Chloramphenicol Stock Solution (100 mg/ml) WARNING: Carcinoge n.
1 g chloramphenicol dissolved in absolute ethanol.
QS to 10 ml with ethanol. Store at -20°C.
CsCl, Isopropanol Equilibrated (1 g/ml in 1x SSPE)
10 ml of 20X SSPE
200 g CsCl
QS to 200 ml with 20X SSPE. Autoclave. After cooling, add 200 ml of isopropyl alcohol, mix well allow to equilibrate overnight before use. (Isopropanol is the top layer).
DEAE Buffers for BAC-Polyacrylamide Gels
100mM NaCl-DEAE Buffer (10mM Tris-HCl, pH 7.9; 1mM EDTA; 0.1 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0.5 M EDTA
10 ml 5 M NaCl -- or -- 50 ml 1.0 M NaCl
pH to 7.9 and QS to 500 ml with water; autoclave.
300 mM NaCl-DEAE Buffer (10 mM Tris-HCl, pH 7.9; 1 mM EDTA; 0.3 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0.5 M EDTA
30 ml 5 M NaCl
pH to 7.9 and QS to 500 ml with water; autoclave.
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1.0 M NaCl-DEAE Buffer (10mM Tris-HCl, pH 7.9; 1mM EDTA; 1.0 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0.5 M EDTA
100 ml 5M NaCl
pH to 7.9 and QS to 500ml with water; autoclave.
100x Denhart's Solution (2% BSA, 2% Ficol, 2% PVP in 3x SSPE)
For 100 ml:
2% (w/v) BSA (Sigma Fraction V)
2% (w/v) Ficol (400,000 mw)
2% (w/v) Polyvinylpyrrolidone (40,000 mw)
Dissolve in 3x SSPE (diluted with autoclaved water) and filter sterilize. This solution may be stored at room temperature, 4° C or -30° C.
Dialysis Membrane Treatment: (5% sodium bicarbonate, 1 mM EDTA) for nucleic acid electroelution.
     700 ml     500 ml     400 ml
NaHCO3      &nb sp;   35 g        25 g         20 g
0.5 M EDTA   1.4 ml     1.0 ml      0.8 ml
Cut lengths of tubing a little longer than the width of an agarose gel. Boil membrane for 20 minutes in a beaker covered with a watch glass. Rinse extensively with autoclaved water before use.
0.5 M EDTA, pH 8.0
For 250 ml:
He at water, add 46.53 g of EDTA, disodium salt (372.24 mw). Cool to room temperature (may use an ice bath) and adjust pH with NaOH. QS to 250 ml with water and autoclave.
ELISA REAGENT RECIPES
Carbonate Coating Buffer, pH 9.6 (0.15 M sodium carbonate, 0.35 M sodium bicarbonate, 0.03 M sodium azide)
3.18 g Na 2CO3
5.86 g NaHCO3
0.4 g NaN3
Adjust pH to 9.6 with HCl. QS to 200 ml with H 2O. Millipore filter (optional).
10x Phosphate Buffered Saline, pH 7.4 (0.2 M phosphate, 1.5 M NaCl)
2.28 g NaH 2PO 4 (mw=120); 0.038M)
     -or- 2.62 g NaH2PO4(H2O) (mw=137.99; 0.038M)
11.5 g Na2HPO4 (mw=141.96; 0.162 M)
43.84 g NaCl
pH to 7.4. QS to 500 ml with water. Dilute 1:10 for final concentration.
PBS-Tween (washing solution) PBS plus 0.05% Tween-20, 0.02% sodium azide)
400 ml 10x PBS
8 ml 10% NaN3 (azide)
2 ml Tween-20
QS with to 4 L with H2O.
ELISA Diluent (1x PBS, 0.05% Tween-20, 0.1% BSA, 0.02% azide)
50 ml 10x PBS
1 ml 10% sodium azide
0.25 ml Tween-20
0.5 g BSA
QS to 500 ml with H2O.
ELISA Conjugate Diluent
PBS, 0.05% Tween-20, 0.1% BSA, 2 µg/ml IgG)
Make above ELISA diluent and add 1 mg/ml Bovine Cohn Fraction II Ig G
QS to 500 ml with H2O.
Blocking Solution (1x PBS with 1% BSA, 0.02% azide)
50 ml 10x PBS
1 ml 10% sodium azide
5 g BSA
QS to 500 ml with H2O
Substrate Buffer, pH 8.6
1.4 g NaHCO3 (0.167M)
0.13 g Na2CO3 (0.012M)
0.02 g MgCl2 (100 µl 1 M stock; 1 mM)
200 µl 10% NaN3 (0.02%)
Add Na2CO3 slowly while stirring. Adjust pH to 8.6 and QS to 100 ml with H2O.
Substrate Solution (1 mg/ml paranitrophenyl phosphate PNPP)
100 mg capsule of PNPP (Sigma 104-100)
100 ml substrate buffer
Store at -20°C in 10 ml aliquots. Keep protected from light
66% Ethanol, 0.9% NaCl
For 30 ml:
4.61 ml of 1M NaCl
19.8 ml of ETOH
5.59 ml of sterile water
Do not autoclave!
70% Ethanol
2210.4 ml 95% ethanol
QS to 3 L with water.
Don't autoclave.
Ethidium Bromide Stock Solution (10mM Tri s-HCl, 1 mM EDTA, 1 mg/ml ethidium bromide)
For 50 ml:
0.5 ml 1M Tris-HCl, pH 8.0
0.1 ml 0.5M EDTA
1 mg ethidium bromide
Add ethidium bromide to Tris-HCl, EDTA and about half of the water, and stir overnight to dissolve. QS to 50 ml with water. Store in a brown bottle at room temperature.
Ethidium Bromide Plates (10mM Tris-HCl, 1 mM EDTA, 0.8% agarose, 2 mg/ml ethidium bromide)
For 1 Liter (approximately 25-30 plates) :
10 ml 1M Tris-HCl, pH 8.0
2 ml 0.5M EDTA
8 g agarose
1 L water
Boil to dissolve agarose. Let cool to about 60°C. Add 2 mg ethidium bromide (400µl of 5 mg/ml stock), mix well and pour plates.
Deionized Formamide
Mix 10 ml formamide with 0.5 g mixed bed resin (Sigma; Amberlite MB-1) gently for 30 minutes at room temperature. Filter through a Whatman #1 filter to remove resin. Aliquot 950 µl into a 1.5 ml microfuge tube to make 1 ml formamide dye mix for sequencing. Store at 4°C.
50 mM Glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA
For 100 ml:
0.90 g glucose
2.50 ml 1 M Tris-HCl
2.0 ml 0.5 M EDTA
Adjust pH to 8.0 with NaOH. QS to 100 ml with autoclaved water and filter sterilize.
30% Glycerol
30 ml 100% glycerol
70 ml water
Autoclave.
50% Glyc erol
25 ml 100% glycerol
25 ml water
Autoclave. Concentrated glycerol is quite viscous. Be sure all of it has been transferred from the stock bottle.
10x H buffer: (66 mM Tris-HCl pH 7.4; 66 mM MgCl2; 66 mM DTT; 0.5M NaCl)
132 µl 500 mM Tris-HCl stock, pH 7.4
  66 µl 1M MgCl2 stock
  ; 66 µl 1M DTT stock
100 µl 5M NaCl stock
QS to 1 ml with H2O
1 M HCl
8.6 ml concentrated HCl
QS to 100 ml with water. Do not autoclave. Wear rubber gloves & open stock bottle of acid in the fume hood.
20X Hellings (1M Tris, 0.4 M sodium acetate, 0.04 M EDTA, pH 8.05)
Chemical For 4 liters For 2 Liters
Tris 484.4   g 242.2 g
Sodium Acetate (anhydrous) 131.25 g   65.6 g
EDTA   59.56 g   27.8 g
pH with glacial acetic acid to 8.05 (try about 60 ml initially, check pH and adjust as needed. QS to 4 liters (or 2 L) with distilled water. Autoclaving is optional.
Hybridization Solution for Cloned DNA (5x SSPE, 10x Denhardt's, 0.1% SDS)
For 100 ml:
25 ml 20x SSPE aut oclaved stock
10 ml 100X Denharts solution, filter sterilized
1 ml 10% SDS stock
Use sterile water. Do not autoclave Filter sterlize.
Hybridization Solution for Genomic DNA (30 ml)
7.5 ml 20x SSPE
1.5 ml 100x Denhardt's Solution
3 ml 50% dextran sulfate
1.5 ml 1M phosphate buffer, pH 6.7
0.3 ml 10% SDS
16.05 ml H2O
Add 150 µl of 10 mg/ml denatured sal mon sperm DNA for pre-hybridization.
IPTG (100mM)
23.8 mg per ml autoclaved water.
Weigh out approximately this amount; note the amount weighed and dissolve in the appropriate volume of autoclaved water. Store at 4°C.
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
L Broth Agar
1 L of L Broth
15 g agar
Autoclave
L Broth Top Agar
Make L Broth Agar except substitute 7.5 g agar/L.
Erythrocyte Lysing Solution
0.83 g NH4Cl
0.1 g KHCO3
90 µl of 10% EDTA (optionally added as a preservative)
QS to 100 ml with water. Filter steri lize, and store at room temperature
1 M Magnesium Chloride
For 50 ml:
10.17 g MgCl2(H2O)6 (i.e. hexahydrate; mw = 203.3)
QS to 50 ml with water and autoclave.
1 M Magnesium Sulfate
For 100 ml:
24.65 g of MgS04(H2O)7 (i.e. heptahydrate; mw = 246.5)
QS to 100 ml with water and autoclave.
Minimal Agar Plates (for 1L)
15 g Difco minimal agar (use Bacto Agar) in 800 ml H2O. Autoclave.
Salts: (makes a 5x solution. QS to 200 ml with H2O and autoclave)
        10.5 g K2HPO4 (13.75 g for K2HPO4-3H2O)
        4.5 g KH2PO4
        1.0 g (NH4)2SO4
        0.5 g sodium citrate(2H2O)
Magnesium sulfate solution:
        Make and autoclave a 20g/100ml stock.
Thiamine-HCl Solution: (Various concentrations may be made)
        1% stock: 0.1 g thiamine-HCl (vitamin B1) to 10 ml water
             -or-
        20% stock: Dissolve 2 mg/ml water
        Autoclave.
Glucose Solution (20% stock):
        Dissolve 20 g glucose in water. QS to 100 ml. Add 10 ml.
Autoclave agar, salt solution, magnesium solution, and nutrients separately. Final concentration of D,L-amino acids is 40 µg/ml, or 20 µg/ml for L-amino acids. Vitamins are usually added to 1 µg/ml. These are then added to the salts after autoclaving. For our plates, we add 1 ml of the Mg stock solution, 25 µl of the 20% thiamine solution (or 0.5 ml of 1% stock), and 10 ml of glucose solution to the salt and agarose solutions. Other additives may be added as needed.
Neutralizing Solution (0.5 M Tris-HC l, 3M NaCl, pH 7.4) for Southern and Benton Davis blots.
For 2 Liters:
121.1 g Tris
350.7 g NaCl
Adjust pH to 7.4 with concentrated HCl (c. 70 ml?) and QS to 2 liters with water.
Nick Translation Elution Buffer (10 mM Tris-HCl, pH 7.5; 50 mM EDTA, 0.2% SDS).
For 100 ml:
1 ml 1.0 M Tris-HCl, pH 7.5
10 ml 0.5 M EDTA
2 ml 10% SDS
Use sterile pipets to transfer solutions to an autoclaved bottle. QS to 100 ml with sterile w ater. No need to autoclave.
10X Nick Translation Buffer(0.5 M Tris-HCl, pH 7.5; 0.1 M MgSO4; 10 mM DTT; 0.5 mg/ml BSA)
For 500 µl:
250 µl 1.0 M Tris-HCl, pH 7.5
50 µl 1.0 M MgSO4
5 µl 1.0 M DTT stock (not autoclaved)
5 µl BSA (BRL 50 mg/ml stock)
190 µl water
Nick Translation Column
5 g Sephadex G-50 fine (medium may be used) stored at room temp in 100 ml of 50 mM EDTA;
10 mM Tris-HCl, pH 7.8; 0.1% sodium azide.

Northern Blot Solutions
5x MOPS Running Buffer(0.2 M MOPS, pH 7.0; 50 mM sodium acetate; 5 mM EDTA)
20.93 g MOPS free acid
2.05 g sodium acetate
5.0 ml 0.5 M EDTA (pH 8.0 stock)
QS to about 475 ml with water. Adjust pH to 7.0 with NaOH. QS to 500 ml with water. Wrap in foil (light sensitive) and autoclave.
Sample Loading Buffer
F or around 20 µg of total cellular RNA or 1-2 µg of poly-A RNA, QS to 5 µl with water. Add 4 µl 5x MOPS running buffer, 3.5 µl formaldehyde and 10 µl of deionized formamide. Heat 10 - 15 minutes at 65°C. Place on ice and add 2 µl of stop dye.
Acridine Orange Stain
Stain the gel in about 300 ml of 40 µg/ml acridine orange dissolved in 10 mM sodium phosphate buffer, pH 6.8

Preparation of Sodium P hosphate Buffers
Prepare a 0.2 M monobasic stock by disolving 27.6 g of monobasic sodium phophate, monohydrate, in water and q.s to 1 liter. Prepare a dibasic 0.2 M stock solution by dissolving 28.4 g dibasic sodium phosphate in water and q.s. to 1 liter. Mix appropriate volumes of monobasic and dibasis sodium phophate stocks and q.s. to 200 ml to obtain a 0.1 M phophate buffer with the desired pH. The table below may also be used for other molar concentrations of phosphate buffer stocks.
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0.1 M Phosphate Buffers
 pH   Monobasic Stock   Dibasic Stock 
5.8
92.0 ml
8.0 ml
5.9
90.0 ml
10.0 ml
6.0
87.7 ml
12.3 ml
6.1
85.0 ml
15.0 ml
6.2
81.5 ml
19.5 ml
6.3
77.7 ml
22.5 ml
6.4
73.5 ml
26.5 ml
6.5
68.5 ml
31.5 ml
6.6
62.5 ml
37.5 ml
6.7
56.5 ml
43.5 ml
6.8
51.0 ml
49.0 ml
6.9
45.0 ml
55.0 ml
7.0
39.0 ml
61.0 ml
7.1
33.0 ml
67.0 ml
7.2
28.0 ml
72.0 ml
7.3
23.0 ml
77.0 ml
7.4
19.0 ml
81.0 ml
7.5
16.0 ml
84.0 ml
7.6
13.0 ml
87.0 ml
7.7
10.5 ml
89.5 ml
7.8
8.5 ml
91.5 ml

10x Phosphate Buffered Saline, pH 7.4 (0.2 M phosphate, 1.5 M NaCl)
2.28 g NaH2PO4 (mw=120 ; 0.038M)
    -or- 2.62 g NaH2PO4(H2O) (mw=137.99; 0.038M)
11.5 g Na2HPO4 (mw= 141.96; 0.162 M)
43.84 g NaCl
Adjust pH to 7.4. QS to 500 ml with water. Dilute 1:10 for final concentration.
20% Polyethylene Glycol, 2.5 M NaCl
25 ml 5 M NaCl
10 g PEG (mw 8000)
QS to 500 ml with H2O and autoclave.
PLASMID PREP SOLUTIONS
Solution #1: 0.1M NaCl; 50mM Tris-HCl pH 7.8; 10mM EDTA
20 ml 5 M NaCl
50 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0
Use HCl to pH to 7.8 (takes approximately 1 ml of concentrated acid).
QS to 1 liter with water and autoclave.
Solution #2: 25% Sucrose in 5 0 mM Tris-HCl pH 8.0
25 ml 1 M Tris-HCl, pH 8.0
125 g sucrose
Adjust pH with NaOH. QS to 500 ml with water and autoclave.
Solution #3: 0.25 M Tris-HCl pH 8.0
For 200 ml:
50 ml 1 M Tris-HCl, pH 8.0
Adjust pH to 8.0. QS to 200 ml with water and autoclave.
Solution # 4: 0.25 M EDTA, pH 8.0
For 250 ml:
125 ml 0.5 M EDTA pH 8.0
pH with NaOH to 8.0. QS to 250 ml with water and autoclave.
So lution #5: 0.3% Triton X-100 in 0.15M Tris-HCl pH 8.0, 0.18 M EDTA
For 250 ml:
37.5 ml 1 M Tris-HCl pH 8.0
90 ml 0.5 M EDTA pH 8.0
7.5 ml 10% Triton-X 100 (v/v)
Adjust pH to 8.0. QS to 250 ml with water and autoclave.
Solution #6: 30% PEG, 1.5M NaCl
150 ml 5 M NaCl
150 g PEG (mw: 6000 or 8000)
QS to about 500 ml with water and autoclave to dissolve the PEG.
After autoclaving. QS as needed with autoclaved H2O to 500 ml.
Solution #7: 50mM Tris-HCl pH 8.0, 50mM NaCl, 5mM EDTA
For 200 ml:
10 ml 1 M Tris-HCl, pH 8.0
2 ml 5 M NaCl
2 ml 0.5 M EDTA, pH 8.0
QS to 200 ml and autoclave.
Solution #8: Ethidium Bromide Solution (5 mg/ml)
For 100 ml:
Dissolve 0.5 g of ethidium bromide with constant stirring in autoclaved water in a sterile brown glass (or foil wrapped) bottle. This may take overnight to dissolve.
Solution #9: Isopropanol-equilibrated Cesium Chloride Solution
Equilibrate an equal volume of isopropanol with 1 g/ml of CsCl in 1x SSPE.

2.7 M Potassium Acetate, pH 4.8
For 50 ml: 13.26 g
60 ml: 15.91 g
100 ml: 26.51 g
Adjust pH with glacial acetic acid. (Approximately 1/2 of the final volume is acetic acid.) The pH & concentration are critical!
< B>1 M Potassium Chloride
For 100 ml:
7.46 g KCl
Dissolve in water and QS to 100 ml with water
Pronase Buffer:
25 mM Tris-HCl, pH 7.5 (1.25 mls of a 1M stock)
0.1 mM EDTA (10 µl of a 0.5M stock)
QS to 50 ml with water and autoclave for 25 minutes, slow exhaust.
Protease Inhibitor Cocktail
Final concentrations are:
1 mM phenylmethylsulfonyl fluoride (PMSF; ma de fresh each week in methanol)
1 mM EDTA
1 mM benzamidime
1 µg/ml pepstatin A
1 µg/ml leupeptin
RBC NH4Cl Lysing Solution
0.83 g NH4Cl
0.1 g KHCO3
90 µl of 10% EDTA
QS to 100 ml with H2O
RNAse (10 mg/ml)
Dissolve RNAse A in water. Boil for 5 minutes. Store at -20° C. Some protocols also add an equal volume of 40% glycerol and a half volume of 0.5 M NaCl.
10% SDS Stock(w/v)
10 g SDS
QS to 100 ml with water. (Autoclaving is optional. We do it).
Sephadex, Equilibration Buffer (50mM EDTA; 10 mM Tris-HCl, pH 7.5; 0.1% sodium azide)
For 1 liter:
100 ml 0.5 M EDT A
20 ml 0.5 M Tris-HCl, pH 7.4
Check pH. QS to 1 liter with water and autoclave
3% Silanizing Solution
15 ml dimethyldichlorosilane
485 ml carbon tetrachloride
Carefully pipet the silane using a glass serological pipet into a glass graduated cylinder containing CCl4. Carbon tetrachloride is toxic! Use fume hood.
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
Sodium Azide, 10%
For 20 ml:
2g sodium azide
QS to 20 ml with water
1 M Sodium Chloride
17.53 g NaCl
QS to 300 ml with water and autoclave.
5 M Sodium Chloride
73.06 g NaCl
Add NaCl gradually to 200 ml H2O. QS to 250 ml with water and a utoclave.
30% Sodium Hydroxide (w/w)
39.9 g NaOH
QS to 100 ml with water. Do not autoclave!
20X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0)
175.4 g NaCl
88.2 g sodium citrate dihydrate
Adjust pH to 7.0 with HCl. QS to 1 L with water. Autoclave.
20X SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.0)
350.7 g NaCl
55.2 g NaH2PO4(H20) (--or-- 48 g anhydrous NaH2PO4, mw = 120.0)
14.89 g EDTA
Dissolve in boiling water, cool to room temperature, & adjust pH to 7.0 with NaOH. QS to 2 liters with water and autoclave.
STE (10 mM Tris-HCl,pH 7.8; 10 mM NaCl; 1 mM EDTA)
Heat water and a dd 0.727 g of Tris base
1.2 ml 5 M NaCl
1.2 ml 0.5 M EDTA
Adjust pH to 7.8. QS to 600 ml with water and autoclave.
STET (50 mM Tris-HCl, pH 8.0; 50 mM EDTA; 8% Sucrose; 5% Triton X-100)
For 500 ml:
3.03 g Tris
9.31 g disodium EDTA
40 g sucrose
25 ml 100% Triton X-100
Dissolve and QS to 500 ml with water. Autoclave.
Stop Dye (0.2% bromophenol blue, 0.2% xylene cyanol, 50% glycerol, 2.5 mM E DTA, pH8.0).
For 100 ml:
0.2 g bromophenol blue
0.2 g xylene cyanol
50 ml glycerol
0.5 ml 0.5 M EDTA, pH 8.0
QS with H2O and autoclave.
Superbroth
33 g tryptone
20 g yeast extract
7.5 g NaCl
1 liter water
Autoclave.
10X TBE (1M Tris, 1M Boric Acid, 20mM EDTA, pH 8.3)
242.2 g Tris
123.66 g boric acid
14.89 g ED TA
Adjust pH to 8.3. QS to 2 liters. Autoclave. (Biotechniques 10:182, 1991 claims that filtering up to a 20x TBE solution through 0.2 - 0.45µ cellulose acetate or cellulose nitrate filters prevents formation of precipitants during long-term storage. The solution may be reautocalved to dissolve precipitates that form.)
Tris-buffered Saline
50 mM Tris-HCl, pH 8.0
150 mM NaCl
T E (10 mM Tris-HCl pH 7.8; 1 mM EDTA)
For 600 ml solution:
0.727 g Tris
1.2 ml of 0.5M EDTA
pH to 7.8 with approximately 7 drops of concentrated HCl with a pasteur pipet (ACID - wear rubber gloves), or about 4 ml of 1 M HCl. QS to 600 ml with water and autoclave.
Tetracycline Stock Solution (20mg/ml; 1000X stock)
0.2 g tetracycline
Dissolve in 10 ml of methanol and store in -30°C freezer in a sterile tube.
Thymidine (4mg/ml)
35.9 mg Thymidine
Dissolve in 8.975 ml water and filter sterilize.
TM Buffer for bacteriophage resuspension
50 mM Tris-HCl, pH 7.8 (25 mls of a 1M stock)
10 mM MgSO4 (5 mls of a 1M stock)
QS to 500 ml with water and autoclave for 25 minutes, slow exhaust.
TMG (10 mM Tris-HCl, pH 7.5; 10 mM MgSO4; 0.1% gelatin)
For 100 ml:
1 ml 1 M Tris-HCl pH 7.5
1 ml 1 M MgS04
0.1 g gelatin
QS to 100 ml with water and autoclave.
TNE (0.1 M NaCl; 10 mM Tris-HCl, pH 8.0; 1 mM EDTA)
For 100 ml:
10 ml 1 M NaCl
1 ml 1 M Tris-HCl, pH 8
0.2 ml 0.5 M EDTA
QS to 100 ml with water and autoclave.
1.0 M Tris-HCl, pH 8.0
121.1 g Tris
pH with concentrated HCl (approximately 47 ml) - wear rubber gloves - ACID. QS to 1 liter with water and autoclave.
0.5 M Tris-HCl Stocks (100 ml)
(pH is as indicated when diluted 1:10 in water to make a 50 mM working concentration)
pH g Tris Base g Tris-HCl
7.2 0.67 7.02
7.4 0.97 6.61
7.8 1.97 5.32
1 M Tris-HCl Solutions
Dissolve 121.1 g tris in about 900 ml of water. Add concentrated HCl to adjust to the desired pH, then QS to 1 liter with water. OR...you may use the following table to obtain the desired pH.

1M Tris Working Solutions from 1M Stock Solutions
Temp / Final pH       Volume to Add for 1 L          
4°C 25°C  37°C ml 1 M Tris base ml 1 M Tris-HCl
7.53 7.00  6.73
82
918
7.58 7.10 6.82
100
900
7.67 7.20 6.94
121
879
7.77 7.30 7.03
154
846
7.87 7.40 7.12
188
812
7.95 7.50 7.21
223
777
8.04 7.60 7.31
261
739
8.13 7.70 7.41
309
691
8.23 7.80 7.50
350
650
8.31 7.90 7.60
398
602
8.39 8.00 7.69
448
552
8.48 8.10 7.79
499
501
8.57 8.20 7.88
551
449
8.66 8.30 7.99
600
400
8.75 8.40 8.10
649
351
8.85 8.50 8.20
698
302
8.96 8.60 8.31
744
256
9.04 8.70 8.40
782
218
9.13 8.80 8.48
817
183
9.22 8.90 8.57
843
157
9.33 9.00 8.67
873
127
9.48 9.10 8.83
906
94
9.59 9.20 8.94
923
77

20 mM Tris-HCl, pH 7.8; 50mM EDTA; 1% SDS
For 100 ml:
10 ml 0.5 M EDTA
20 ml 0.5 M Tris-HCl, pH 8.0
10ml 10% SDS solution
QS to 100ml with water and autoclave.
Tris-HCl pH 7.8; 10mM NaCl; 0.2mM EDTA
For 1 liter:
10 ml 1 M NaCl
0.4 ml 0.5 M EDTA
20 ml 1 M Tris-HCl, pH 7.8
Adjust pH. QS to 1 liter with water and autoclave.
Triton-X 100
45 ml water 5 ml Triton-X 100 (100% solution)
Autoclave.
Trypan Blue (10X Stock; 0.4%)
200 mg trypan blue
40 ml PBS, pH 7.2 - 7.4
Bring to boil to dissolve, cool, and QS to 50 ml with PBS.
To determine cell viability, add 0.1 volumes of trypan blue solution to 0.9 volumes of cell suspension. Unstained cells are viable.
WESTERN BLOT SOLUTIONS
Lysate Buffer (0.1 M NaCl; 50 mM Tris-HCl, pH 7.8-8.5; 10 mM EDTA)
For 100 ml:
2 ml 5 M NaCl
5 ml 1 M Tris-HCl stock at the appropriate pH
2 ml 0.5 M EDTA
QS with distilled water and autoclave.
5% Nonfat Dry Milk
Prepare using Carnation Nonfat Dry Milk in TBST.
TBST (10 mM Tris-HCl, 0.15 M NaCl, 8 mM sodium azide, 0.05% tween-20, pH 8.0)
500 µl 10% NaN3
Chemical For 4 liters For 5 00 ml
Tris 4.84 g 0.61 g Tris
NaCl 35.06 g 4.38 g NaCl
NaN3 2.0 g
Tween-20 2.0 ml 250 µl Tween-20
Adjust pH to 8.0 with HCl. QS with water and store at 4°C.
Tank Buffer (25 mM Tris-HCl, 0.2 M glycine, 0.35% SDS)
Chemical For 4 liters For 500 ml
Tris 12.11g 1.51 g Tris
Glycine 57.6 g 7.2 g
SDS 4.0 g 17.5 ml 10% SDS
QS with H2O. pH should be 8.3 but adjusting is not needed. Store at 4°C
Transfer Buffers:
Wet Blot Transfer Buffer (3L)   (25 mM Tris-HCl, 0.2 M glycine, 20% methanol).
9.08 g Tris
43.24 g glycine
600 ml methanol
QS to 3 L with water.
Dry Blot Transfer Buffers
Stock Solution A1 (3 M Tris)
36.3 g Tris, dissolved in double distilled water and QS to 100 ml
Store at room temperature.
Stock Solution A2 (0.3 M Tris)
3.63 g Tris, dissolved in double distilled water and QS to 100 ml
Store at 4°C.
Stock Solution C (0.3 M Tris, 0.4 M aminohexane)
3.63 g Tris, 5.2 g aminohexane, dissolved in double distilled water and qs to to 100 ml.
Store at 4°C.
Dry Blot Working Solutions (made f rom fry blot transfer buffers)
7 ml H20
2 ml Methanol
1 ml stock solution of A1, or A2 or C
Store at 4°C.
Lower Gel Buffer (1.5 M Tris-HCl, 0.4% SDS, pH 8.8)
For 100 ml:
18.17 g Tris
4 ml 10% SDS
Adjust pH to 8.8 with HCl. QS to 100 ml with water. Store at room temperature.
Upper Gel Buffer 0.5 M Tris-HCl, 0.4% SDS, pH 6.8)
For 100 ml:
6.06 g Tris
4 ml 10% SDS
Adjust pH to 6.8 with HCl. QS to 100 ml with water. Store at room temperature.
2x Sample Buffer (Hoeffer Scientific)  (125 mM Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue).
Prepare in fume hood!
2.5 ml 0.5 M Tris-HCl, pH6.8 and 4.0 ml of 10% SDS
    (--or-- 2.5 ml of upper gel buffer and 3.6 ml of 10% SDS)
2.0 ml glycerol
1.0 ml concentrated 2-merca ptoethanol
0.4 mg bromophenol blue (you may use less but don't add more than 0.4 mg)
QS to 10 ml with water. Transfer to 1 ml aliquots and store at -20°C.
Stock Sample Buffer (from Craig Wasson) (0.312 M Tris-HCl, 0.346 M SDS, 50% glycerol, pH 6.8)
3.03 g Tris
8.0 g SDS
40 ml glycerol
Adjust pH to 6.8 with HCl. QS to 80 ml with water. Store at room temperature.
4x Sample Buffer (made from stock sample buffer) (0.24 M Tris-HCl, 0.24 M SDS, 40% glycerol, 20% 2-mercaptoethanol, pH 6.8)
8 ml of stock sample buffer
2 ml of concentrated 2-mercaptoethanol
Transfer to 1 ml aliquots and store at -20°C.
0.1% Fast Green (in 10% acetic acid and 20% methanol)
0.2 g Fast Green
20 ml acetic acid
40 ml methanol
140 ml double distilled water.
0.1% Amido Black (in 10% acetic acid and 45% methanol)
0.2 g Buffalo Black
90 ml methan ol
20 ml acetic acid
90 ml double distilled water.
Amido Black Destain (2% acetic acid, 45% methanol)
225 ml methanol
10 ml acetic acid
QS to 500 ml with double distilled water.
0.2% Coomassie Gel Stain (in 7.5% acetic acid and 40% methanol)
0.2 g Coomassie
7.5 ml acetic acid
40 ml methanol
QS with water to 100 ml. Filter (Nalgene or Whatman) before use.
Coomasie Rapid Destain (7.5% acetic acid and 40% methanol)
75 ml acetic Acid
400 ml methanol
QS with water to 1 L. Destain gel about 1 hour while shaking. Change the destaining solution every 15 minutes. Destaining may be done overnight. Dry gel for 1 hour on gel dryer.
Xgal (4%)
50mg 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (Xgal) in 1.25 ml dimethylformamide.
Weight out approximately this amount, then dete rmine the volume of solvent needed for final concentration. Light sensitive - Keep in foil wrapped container. The solution should be clear. Discard if yellow-brown or pink.

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
 Proper citation for data acquired from this document is: "Frank, M. B. Laboratory Solutions. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/recipes.html). 199 7. Oklahoma City. Revision Date: October 2, 1997."