Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

Common Stock Solutions

10 M Ammonium Acetate

To prepare a 10 M solution in 100 ml, dissolve 77 g of ammonium acetate in 70 ml of H₂O at room temperature. To prepare a 5 M solution in 100 ml, dissolve 38.5 g in 70 ml of H₂O. Adjust the volume to 100 ml with H₂O. Sterilize the solution by passing it through a 0.22μm filter. Store the solution in tightly sealed bottles at 4 ° C or at room temperature. Ammonium acetate decomposes in hot H₂O and solutions containing it should not be autoclaved.

Ampicillin

Prepare a stock of 100 mg/ml in water. Sterilize by filtration. Store at -20°C but avoid repeated freeze/thaw cycles. Use at a final concentration of 100 µg/ml.

Cresol Red Loading Dye - 2.5X - for PCR reactions

EB buffer (Qiagen recipe)

10 mM Tris-Cl pH 8.3

EDTA stock

To prepare 1 liter, 0.5M EDTA pH 8.0: Add 186.1 g of disodium EDTA-2H 2O to 800 ml of H 2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH. For tetrasodium EDTA, use 226.1 g of EDTA and adjust pH with HCl.

6x gel loading buffer

0.25% Bromophenol blue

0.25%Xylene cyanol FF

15% Ficoll Type 4000

120 mM EDTA

IPTG

IPTG is isopropylthio-b-D-galactoside. Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled H 2O. Adjust the volume of the solution to 10 ml with H 2O and sterilize by passing it through a 0.22μm disposable filter. Dispense the solution into 1 ml aliquots and store them at -20 ° C.

LB Medium

To make 1 liter, use 10 g tryptone, 5 g yeast extract, 10 g NaCl. Adjust pH to 7.0. Sterilize by autoclaving.

LB Agar

Dispense 15 g per liter of agar directly into final vessel. Prepare LB medium as above and add to agar. NOTE: Agar will not go into solution until it is autoclaved (or boiled). If adding antibiotics, autoclave medium first and allow to cool until warm to the touch, then add the antibiotic. Dispense about 30 ml per plate. Allow plates to dry either at 37°C overnight or 20 minutes in a laminar flow hood (lids removed). Store in original Petri plate bags, inverted, at 4°C for up to 2 weeks.

NaCl

To prepare 1 liter of a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H 2O. Adjust the volume to 1 liter with H 2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.

NaOH

The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause breakage of glass containers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2O. Store the solution in a plastic container at room temperature. Sterilization is not necessary.

20X SB (electrophoresis buffer)

(Buffer diluted to 1X should be 10 mM Sodium hydroxide and pH 8.5 )

for 1 liter, weigh out 8 g NaOH and ~40 g boric acid - add water, dissolve and add additional boric acid until pH = 8.0; bring final volume to 1 liter.

SDS stock

10% or 20% (w/v) SDS. Also called sodium lauryl (or dodecyl) sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-grade SDS in 900 ml of H₂O. Heat to 68 ° C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 1 liter with H 2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.

Use a mask when weighing this out.

20x SSC

0.3M Na(3) citrate

3M NaCl

SOB Medium: per liter:

Bacto-tryptone 20 g

Yeast extract 5 g

NaCl 0.584 g

KCl 0.186 g

Mix components and adjust pH to 7.0 with NaOH and autoclave.

2 M Mg ++ stock:

MgCl 2-6H2O 20.33 g

MgSO 4 -7H2O 24.65 g

Distilled water to 100 ml. Autoclave or filter sterilize.

2 M Glucose

Glucose 36.04 g

Distilled water to 100 ml. Filter sterilize.

For SOB Medium + magnesium: Add 1 ml of 2 M Mg ++ stock to 99 ml SOB Medium.

For SOCMedium: Add 1 ml of 2 M Mg ++ stock and 1 ml of 2 M Glucose to 98 ml of SOB Medium.

3M Sodium Acetate - pH 5.2

Southern Solutions:

Depurination solution (for Southern blotting)

250mM HCl

Denaturation solution (for Southern blotting)

1.5M NaCl

0.5M NaOH

Neutralization solution (for Southern blotting)

1.5M NaCl

0.5M Tris-HCl, pH adjusted to 7.5

50x TAE

Prepare a 50x stock solution in 1 liter of H₂O:

242 g of Tris base

57.1 ml of glacial acetic acid

100 ml of 0.5 M EDTA (pH 8.0)

The 1x working solution is 40 mM Tris-acetate/1 mM EDTA.

5X (or 10X) TBE

Prepare a 5x stock solution in 1 liter of H2O:

54 g of Tris base

27.5 g of boric acid

20 ml of 0.5 M EDTA (pH 8.0)

The pH of the concentrated stock buffer should be approx. 8.3.. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more stable because the solutes do not precipitate during storage. Passing the 5x or 10x buffer stocks through a 0.22μm filter can prevent or delay formation of precipitates.

TE buffer:

10 mM Tris-Cl (pH, usually 7.6 or 8.0)

1 mM EDTA (pH 8.0)

Use concentrated stock solutions to prepare. If sterile water and sterile stocks are used, there is no need to autoclave. Otherwise, sterilize solutions by autoclaving for 20 minutes. Store the buffer at room temperature.

1 M Tris-Cl – used at various pHs

Using Tris base : To make 1 liter, dissolve 121 g Tris Base in 800 ml of water. Adjust pH to the desired value by adding approximately the following:

Make sure solution is at room temperature before making final pH adjustments. Bring final volume to 1 liter. Sterilize by autoclaving.

Using Trizma tables: an alternate procedure for preparing Tris solutions is to combine the proper amount of Tris Base and Tris Hydrochloride to achieve the desired value using Sigma's Tris tables.

WB (10% redistilled glycerol, 90% distilled water, v/v)

Western Blotting Solutions:

1X Transfer buffer 1: 25 mM Tris, 192 mM Glycine, pH 8.3

Mix 3.03 g Tris, 14.4 g glycine; add dd water to 1 liter – do not adjust pH.

1X Transfer buffer 2: 25 mM Tris, 192 mM Glycine, pH 8.3, 20 % methanol

Mix 3.03 g Tris, 14.4 g glycine; add 200 ml methanol; add dd water to 1 liter – do not adjust pH. (NOTE: methanol is not needed for PVDF membranes)

10X Western Buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl

To prepare 1X Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove 50 ml and set aside for the last two washes. To the remainder, add I-Block to 0.2%, heating gently with constant stirring until dissolved. Bring to room temperature before using.

X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactoside (same recipe for X-phosphate)

Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to prevent damage by light and store at -20 ° C. It is not necessary to sterilize X-gal solutions.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 1/12/2006