BioProtocols--msol5

Solutions for molecular cloning--1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )

Lambda top agar:

10 g Bacto-tryptone (Difco 0123-01-1)

10 g Bacto-agar (Difco 0140-01)

5 g NaCl

ddH2O to 1 L

autoclave to sterilize

LB Medium:

10 g Bacto-Tryptone (Difco 0123-01-1)

5 g Bacto-yeast extract (Difco 0127-05-3)

10 g NaCl

ddH2O to 1 L

adjust the pH to 7.0 and then

autoclave to sterilize

(p> LB plates:

10 g Bacto-Tryptone (Difco 0123-01-1)

5 g Bacto-yeast extract (Difco 0127-05-3)

10 g NaCl

15 g Bacto-agar (Difco 0140-01)

ddH2O to 1 L

autoclave to sterilize, cool to 55deg.C, add antibiotic if desired, and pour into sterile petri dishes (approx. 20 ml/plate).

10x Ligation buffer: 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 10 mM DTT, 10 mM rATP, and 250 ug/ml bovine serum albumin (BSA) in sterile double distilled water.

5 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl2

1 ml 1 M DTT

1 ml 100 mM rATP

2.5 mg BSA

sddH2O to 10 ml (store in 25 ml aliquots at -20deg.C)

Loading dye: 0.3% xylene cyanole FF, 0.3% bromophenol blue, 10 mM EDTA in deionized formamide.

3 g xylene cyanole FF

3 g bromophenol blue

0.2 ml 0.5 M EDTA

deionized formamide to 10 ml

Lysozyme solution: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 5 mg/ml lysozyme in sterile double distilled water.

5 ml 1 M Tris-HCl, pH 8.0

2 ml 0.5 M EDTA

0.5 g lysozyme (Sigma L-6876)

sddH2O to 100 ml (make fresh)

M-9 Agar: Add 15 g. agar to 870 ml. double distilled water in a 2 L Ehrlenmeyer flask and autoclave. Also autoclave a 100 ml. graduate cylinder capped with aluminum foil to use for measuring the sterile M-9 salts later. Swirl the agar gently and carefully upon removal from autoclave to disperse any undissolved agar. Allow to cool in a 55degC water bath. When 55degC, add the ingredients called for in the M-9 liquid medium recipe, omitting the water. Be sure to use sterile pipettes or graduate cylinders, as this mixture cannot be autoclaved. Immediately pour into sterile Petri dishes, using sterile technique.

M-9 Medium (liquid):

100 ml 10X M-9 salts

1 ml 1 M MgSO4 (autoclaved)

10 ml 20% glucose (filter sterilized),

1 ml 1% thiamine (filter sterilized)

10 ml 100 mM CaCl2 (autoclaved)

sddH2O to 1 L

10X M-9 Salts:

60 g Na2HPO4 (sodium phosphate, dibasic)

30 g KH2PO4 (potassium phosphate, monobasic)

5 g NaCl

10 g NH4Cl (ammonium chloride)

ddH2O to 1 liter (autoclave)

1 M MgCl2 (magnesium chloride):

20.33 g MgCl2-6H2O

ddH2O to 100 ml

1 M MgSO4 (magnesium sulfate):

12.04 g MgSO4

ddH2O to 100 ml (autoclave)

1 M MnCl2 (manganese chloride):

1.98 g MnCl2 (Sigma M-8530)

ddH2O to 10 ml (store protected

from light)

Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )

Commonly used solutions (2)--- 1 ) 2 ) 3 )

Solutions for molecular cloning---1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )