106754587
for Biol 305 fall 2004
October 6, 2004
Boss14
Appendix A: Preparation of Reagents
Note: All reagents are made with double distilled H2O unless otherwise noted.
Stock solutions
1 M Tris
0.5 M EDTA
5 M NaCl
10 M NaOH
SDS (10% w/v)
1 M CaCl22H20
for competent cell
production
Dissolve 121.1 g of Tris-Cl in 800 ml of H20. Adjust the pH to the
desired value by adding concentrated HCl.
desired pH
amount of HCl
7.4
70 ml
7.6
60 ml
8.0
42 ml
Check pH using a pH meter with a Tris compatible electrode, and
adjust. Bring volume to 1 L. Dispense in 100 ml aliquots and
autoclave for 15 min at 15 psi on liquid cycle.
Add 186.1 g of disodium EDTA2H20 to 800 ml of H20. Add 20 g of
NaOH pellets. Stir until all the NaOH has dissolved (EDTA will not
go into solution until pH is close to 8.0). Adjust the pH to 8.0.
Dispense into 100 ml aliquots and autoclave for 15 min at 15 psi on
liquid cycle.
Dissolve 292 g of NaCl in 800 ml of H20. Adjust volume to 1 liter
with H20. Dispense into 100 ml aliquots and autoclave for 15 min at
15 psi on liquid cycle.
Add 40 g of NaOH slowly to 80 ml of H20. Stir until completely
dissolved. Bring volume to 100 ml and store in a plastic bottle.
There is no need to autoclave.
NOTE: This is a exothermic reaction, the beaker will become quite
hot and can break. Use plastic beakers as a precaution.
Dissolve 100 g of SDS in 900 ml of H20. Heat gently and stir to add
dissolution. Dispense into 100 ml aliquots and store at RT. Do not
autoclave.
Dissolve 14.7 g of CaCl22H20 in 80 ml of H20. Bring to volume,
Filter sterilize. Dispense in 10 ml aliquots and store at –20. For
transformation prepare 0.1 M CaCl2 fresh by thawing a 10 ml aliquot
of 1 M CaCl2, adding 90 ml of dd H20 and filter sterilizing.
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106754587
for Biol 305 fall 2004
October 6, 2004
Boss14
Solutions for Alkaline Lysis
Alkaline lysis solution I
final [component]
stock concentration
50 mM glucose
1 M glucose
25 mM Tris-Cl (pH 8.0)
1 M Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
0.5 M EDTA (pH 8.0)
H20
Autoclave for 15 min at 15 psi on liquid cycle.
volume used
to make 100 ml
5 ml
10 ml
2 ml
83 ml
Alkaline lysis solution II
volume used
final [component]
stock concentration
to make 10 ml
0.2 N NaOH
10 N NaOH
200 l
1% SDS
10 % SDS
1 ml
H20
8.8 ml
This is made fresh immediately before use. Do not autoclave.
Alkaline lysis solution III
volume used
component
to make 100 ml
5 M potassium acetate
60.0 ml
glacial acetic acid
11.5 ml
H20
28.5 ml
Dispense into 100 ml aliquots and autoclave for 15 min at 15 psi on liquid cycle.
STE
final [component]
stock concentration
10 mM Tris-Cl (pH 8.0)
1 M Tris-Cl (pH 8.0)
0.1 M NaCl
5 M NaCl
1 mM EDTA (pH 8.0)
0.5 M EDTA (pH 8.0)
H20
Autoclave for 15 min at 15 psi on liquid cycle.
volume used
to make 100 ml
1 ml
2 ml
0.2 ml
97 ml
TE
final [component]
10 mM Tris-Cl (pH 8.0)
1 mM EDTA (pH 8.0)
H20
stock concentration
1 M Tris-Cl (pH 8.0)
0.5 M EDTA (pH 8.0)
volume used
to make 100 ml
1 ml
0.2 ml
97 ml
Page 2 of 4
106754587
for Biol 305 fall 2004
October 6, 2004
Boss14
Solutions for agarose gel electrophoresis.
Gel running buffers1
50X TAE (Tris-acetate) stock
5X TBE (Tris-borate)stock
(1X= 40 mM Tris base, 40 mM Acetic acid,
1 mM EDTA)
(1X=89 mM Tris base, 89 mM Boric acid,
2 mM EDTA)
242.0 g Tris base
54.0 g Tris base
57.1 mL Glacial acetic acid
27.5 g Boric acid
18.61 g Na2 EDTA·2H2O
3.72 g Na2 EDTA·2H2O
To 1 liter with 18  water
To 1 liter with 18  water
1. From Sambrook and Russell 2001.
Loading buffer recipes
Loading Buffer
Sucrose Based
Glycerol Based
Ficoll® Based
6X Recipe
40% (w/v) Sucrose
0.25% Bromophenol Blue
0.25% Xylene cyanol FF
30% Glycerol in distilled water
0.25% Bromophenol Blue
0.25% Xylene cyanol FF
15% Ficoll (type 400) polymer in
distilled water
0.25% Bromophenol Blue
0.25% Xylene cyanol FF
Storage Temperature
4° C
4° C
Room Temperature
Page 3 of 4
106754587
for Biol 305 fall 2004
October 6, 2004
Boss14
Media and antibiotics
LB-broth (Lauria-Bertani Broth)
Place a magnetic stir bar in a 1 L flask. Weigh and add the following:
 10 g NaCl
 10 g tryptone
 5 g yeast
 800 ml of H20
Stir until components have dissolved.
Adjust the pH to 7.0 with 5 N NaOH
Adjust the volume to 1 L.
Transfer to appropriate containers for sterilizing by autoclaving.
SOB media
Per liter:
 20 g tryptone
 5 g yeast extract
 0.5 g NaCl
 800 ml H2O
Dissolve solutes. Add 10 ml of 250 mM KCl (made by dissolving 1.86 g of KCl in 100 ml
H2O). Adjust pH to 7.0. Bring the volume to 1 L. Sterilize by autoclaving.
Just before use add 5 ml of sterile 2 M MgCl2 per L (made by dissolving 19 g of MgCl2
in a total volume of 100 ml, and sterilized by autoclaving).
SOC media
SOB media with 20 mM glucose.
After autoclaving SOB media, cool to 60 ºC or less. Add 20 ml of sterile 1 M glucose (18
g of glucose in a total volume of 100 ml, filter sterilized).
To make agar plates from any media
Follow the procedure for making the liquid media.
After dispensing into desired containers add agar at the rate of 15 g/L.
Sterilize by autoclaving, (agar does not dissolve until heating).
Add antibiotics as required.
Pour approximately 20 ml per plate.
Antibiotics
name
stock concentration
working concentration
Ampicillin
50 mg/ml in H2O
20 – 50 μg/ml
Kanamycin
10 mg/ml in H2O
10 – 50 μg/ml
Tetracycline
5 mg/ml in ethanol
10 – 50 μg/ml
Sterilize by filtering through a 0.22 μM filter, except if dissolved in ethanol.
Before adding antibiotics to media, let the media cool to 50 ºC.
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