Commonly Used Buffers and Solutions

Hongwei Cheng 6/10/01, Edited by TCH 6/28/13

 

 

BD-I Solution for Alkaline Lysis DNA Minipreps

50mM glucose ; 25mM Tris-Cl (pH 8.0) ; 10mM EDTA (pH 8.0)

For 500ml BD-I

Glucose: 4.5g

1M Tris-HCl pH8.0 12.5ml

0.5M EDTA pH8.0 10.0ml

Add ddH2O to 500ml

Filter sterilize

Store at 4oC

 

BD-II for Alkaline Lysis DNA Minipreps

ddH2O 93ml

10M NaOH 2ml

20% SDS 5ml

 

BD-III for Alkaline Lysis DNA Minipreps

5M potassium acetate 60ml

Glacial acetic acid 11.5ml

Adjust pH to 4.8 (at least <5.4) with HCl (approx. 12ml concentrated HCl)

Add ddH2O to 100ml

or

For 100 ml

Potassium acetate: 29.4 g

dH2O: 40 ml

Glacial acetic acid: 11.5 ml

Adjust pH to 4.8 (at least <pH 5.2) with HCl (approx. 12ml concentrated HCl)

dH2O to 100 ml

Filter sterilize

Store at 4oC

 

25x TAE (per liter)

Tris base 121g

Glacial acetic acid 28.55ml

0.5M EDTA (pH 8.0) 50ml

Add ddH2O to 1000ml

 

10x TBE (per liter)

Tris base 108g

Boric acid 55g

0.5M EDTA (pH 8.0) 20ml

Add ddH2O to 1000ml

 

10 M Ammonium Acetate (NH4C2H3O2)

  1. Dissolve 771g ammonium acetate (m.w. = 77.1 g/mole) in 800 ml of H2O
  2. Make final volume of 1 L with H2O
  3. Sterilize by filtration and store at room temperature

 

1 M Dithiothreitol (DTT)

  1. Dissolve 1.5g of DTT (DL-dithiothreitol, anhydrous m.w.=154.25) in 8 ml of deionized or distilled H20
  2. Adjust volume to 10 ml and dispence into 1 ml aliquots
  3. Store in dark, wrapped in foil at 20 C

 

0.5 EDTA (Disodium Ethylene Diamine Tetra-Acetate, pH 8.0) 500mM solution

  1. Add 181.6 g of Na2EDTA2H2O to 800 ml H20
  2. While stirring with a magnetic stirrer, adjust to pH 8.0 with pellets of NaOH (about 20 g NaOH
  3. Sterilize by autoclaving and store at room temperature

 

Isopropyl-Beta-D-Thiogalactopyranoside (IPTG)

  1. Dissolve 2 g of IPTG (m.w. = 238.3 g/mole) in 8 ml of distilled H2O
  2. Adjust to10 ml with H20 and sterilize by filtration.
  3. Dispense the solution into 1 ml aliquots and store at 20 C.

 

1 M Tris with Various pH Values

  1. Dissolve 121.1 g of TRIS base in 800 ml H20
  2. Adjust pH to the desired value by adding concentrated HCl.
  3. Allow solution to cool to room temperature before making final adjustments to the ph
  4. Adjust to 1 L with H20, dispense into, aliquots, sterile by autoclave, and store at room temperature

pH

HCL

7.4

70 ml

7.6

60 ml

8.0

42 ml

 

HEPES Bufffer (For 50 mM HEPES buffer @ pH 7.0)

  1. Mix 25 mL of 200 mM HEPES (52.06 g/liter of HEPES Na salt), 11.8 of 100 mM NaOH
  2. Add H2O to reach 100ml
  3. Sterilize the solution by filtration

 

Phosphate Buffered Saline (PBS) (pH 7.2-7.4)

  1. Dissolve the components in approximately 0.9 liters of H2O
  2. Adjust pH to 7.2-7.4 with HCl and then adjust the final volume to 1 liter with H2O
  3. Sterilize by autoclave for 20 minutes at 15 psi on liquid cycle

Component and final concentration

Amount to add per 1 liter

137 mM NaCl

8 g

2.7 mM KCl

200 mg

10 mM Na2HPO4O (dibasic, anhydrous)

1.44 g

2 mM KH2PO4O (monobasic, anhydrous)

240 mg

H20

To make 1 liter

 

 

Tris-EDTA (TE) (pH 7.4-8.0)

 

This standard buffer is used to resuspend and store DNA. It can be prepared by using stock solutions of 1 M Tris HCL at pH values ranging from 7.4 to 8.0. Store at room temperature

Component and final concentration

Amount of stock to add per 100 ml

10 mM Tris-HCl

1 ml of 1 M (pH 7.4-8.0 at 25 C

1mM EDTA

200ml of 0.5 M (pH 8.0)

H20

98.9 ml

 

10X TE, pH 8 (100mM Tris-HCl, 10mM EDTA

 

Final (1X)

Mix for 10X

Tris-HCl (pH 8.0)

10 mM

100ml of 1M

EDTA

1 mM

20 ml of 500 mM (pH 8.0)

Distilled H2O to 1 liter Sterilize by autoclaving. Store at room temperature

 

Proteins, Enzymes, and Antibiotics

 

20mg/ml Proteinase K

  1. Add 200 mg of proteinase K to 15-ml polypropylene tube containing 9.5 ml H2O
  2. To reduce denaturation, always add protein to an aqueous solution instead of adding aqueous solution to protein.
  3. Gently rock until completely dissolved (no vortexing)
  4. Adjust final volume to 10ml

 

10mg/ml Rnase A (Dnase-free)

  1. Dissolve 10 mg of pancreatic Rnase A in 1 ml of 10mM sodium acetate (pH 5.0)
  2. Place in boiling-water bath for 15 minutes to inactivate any contaminating Dnase
  3. Adjust pH to 7.5 with 1 M Tris-HCl
  4. When cool, aliquot and store at 20 C

 

Electrophoresis of DNA in Agarose Gels

 

50X TAE Buffer

This buffer does not have the buffering capacity of TBE buffer. The 1x TAE buffer (pH 8.1) is 40 mM Tris, 20 mM acetate, and 2 mM EDTA

Component and final concentration

Amount to add per 1 liter

2 M Tris base

242 g

1 M acetate

57.1 ml of glacial acetate acid (17.4 M)

100 mM EDTA

200ml of 0.5 M (pH 8.0)

H2O

To make 1 liter

 

5X TBE Buffer

TBE can be prepared as a 5X or 10X stock buffer, but the 10X stock buffer will precipitate during storage. Store at room temperature. 1X buffer (pH 8.3) is 89 mM Tris, 89 mM borate, and 2 mM EDTA.

Component and final concentration

Amount to add per 1 liter

445 mM Tris base

54 g

445 mM borate

27.5 g of boric acid

10 mM EDTA

20 ml of 0.5 M (pH 8.)

H2O

To make 1 liter

 

 

Sample Buffers for Protein Electrophoresis

2X Laemmli Sample Buffer Stock

  1. Add 4 ml of 10% SDS, 2 ml of glycerol, and 1.2 ml of 1 M Tris (pH 6.8) to 2.8 ml of distilled H2O
  2. Add bromophenol blue to 0.01% as a tracking dye.
  3. Store at room temperature

 

 

20X SSC (Saline Sodium Citrate)

  1. Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2o
  2. Adjust pH to 7.0 with a few drops of a 10 N solution of NaOH
  3. Adjust volume to 1 liter with H2O
  4. Dispense in aliquots and autoclave.

 

20X SSPE (Saline, Sodium Phosphate, EDTA)

  1. Dissolve 175.3 g of NaCl, 27.6 g NaH2PO4H2O, and 7.4 g of EDTA in 800 ml in H20
  2. Adjust pH to 7.4 with NaOH (about 6.5 ml of a 10N solution
  3. Adjust volume to 1 liter with H20
  4. Dispense in aliquots and autoclave

 

 

GSB (6x DNA gel sample buffer)

 

 

SDS-PAGE Sample Buffer (for protein gels)

 

RNA Loading Solution

 

10x PCR Buffer (DMSO protocol)