|20% SDS||LB Freezing Buffer|
|6X Gel loading Buffer||Neutralizing Solution|
Tris-Cl (1 M)
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl <!>.
7.4 70 ml
7.6 60 ml
8.0 42 ml ßour most common
Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.
If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature dependent, and decreases ~0.03 pH units for each 1 C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5 C, 25 C, and 37 C, respectively.
Dissolve 525.9 g of NaCl and 264.6 g of sodium citrate in 2.4 liters of H2O. Adjust the pH to 7 with a few drops of a 14 N solution of HCl <!>. Adjust the volume to 3 liters with H2O. Dispense into aliquots. Sterilize by autoclaving. The final concentrations of the ingredients are 3.0 M NaCl and 0.3 sodium citrate.
Extraction/Lysis Buffers and Solutions
Alkaline Lysis Solution I (Plasmid Preparation)
50 mM glucose
25 mM Tris-Cl (pH 8.0)
10mM EDTA (pH 8.0)
Prepare solution I from standard stocks in batches of ~500 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4 C.
Alkaline Lysis Solution II (Plasmid Preparation)
5 M potassium acetate 180.0 ml
glacial acetic acid <!> 34.5 ml
H2O 85.5 ml
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4 C and transfer it to an ice bucket just before use.
Electrophoresis and Gel-loading Buffers
TAE 50 X (stock solution/Liter)
242 g of Tris base
57.1 ml of glacial acetic acid <!>
100 ml of 0.5 M EDTA (pH 8.0)
TBE 5X (3.5 Liters)
189 g of Tris base
96.25 g of boric acid
70 ml of 0.5 M EDTA (pH 8.0)
LB Freezing Buffer
36 mM K2HPO4 (anhydrous)
13.2 mM KH2PO4
1.7 mM sodium citrate
0.4 mM MgSO4*7H2O
6.8 mM ammonium sulfate
4.4% (v/v) glycerol
100 ml LB broth
Dissolve the salts into 100ml of LB to the specified concentrations. Measure 95.6 ml of the resulting solution into a fresh container and then add the 4.4 ml of glycerol. Mix the solution well and then sterilize by passing it through a 0.45-ul disposable Nalgene filter. Store the sterile freezing medium at a controlled room temperature (15-25 C).
EDTA (0.5 M, pH 8.0)
Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~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 ~8.0 by the addition of NaOH.
NaCl (Sodium Chloride, 5 M)
Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.
SDS (20% w/v)
Also called sodium lauryl sulfate. Dissolve 200 g of electrophoresis –grade <!> 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 H2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.
Sodium Acetate (3 M, pH 5.2 and pH 7.0)
Dissolve 102.07 g of sodium acetate*3H2O in 200 ml of H2O. Adjust the pH with glacial acetic acid <!> or adjust the pH to 7.0 with dilute acetic acid. Adjust the volume to 250 ml with H2O. Dispense into aliquots and sterilize by autoclaving.
5X Denaturing Solution
bring to 3.5L H2O
CTAB =1% mixed alkyl trimethyl-ammonium bromide
DNA Wash Solution
76% ethanol ____________ 304 ml of 100% ethanol
10 mM NH4Ac _________ 0.4 ml of 10 M NH4Ac
Bring volume to 400 ml with ddH2O.
0.25% (W/V) Bromophenol blue
0.25% (W/V) Xylene cyanol FF
30% (V/V) Glycerol in H2O store at 4C