Buffers and stocks
TEB (T antigen extraction buffer) [1 % NP-40, 10 % glycerol, 137 mM NaCl, 20 mM
Tris pH7.5]
50 % glycerol
20 ml
5M NaCl
2.74 ml
1M Tris pH 7.5
2 ml
NP-40
1 ml
Water
up to 100 ml final volume
20xTBE (Tris-Borate-EDTA)
2l
0.5 l
Tris- Base
432 g
60.5 g
Boric acid
220 g
30.9 g
0.5M EDTA pH 8.0
160 ml
3.7 g solid
50xTAE (Tris-acetate-EDTA)
0.25 l
Tris-Base
60.5 g
Glacial acetic acid
14.3 ml
0.5 M EDTA pH 8.0
25 ml
0.5 M EDTA pH8.0
0.5 l
EDTA, 2H2O
93.05 g
Water
400 ml
NaOH, pellets
appr. 10 g
Adjust pH to 8.0, then finally volume to 0.5 l. EDTA will not go into solution until pH
approaches 8.0
1M Tris pH 8.0
0.5 l
Tris-base
60.5 g
Adjust pH to 8.0 with conc. HCl then volume
5M NaCl
0.1 l
0.5 l
NaCl
29.2 g
146 g
Water
80 ml
400 ml
When all NaCl is dissolved, adjust volume
10x Running buffer (SDS-PAGE) 1 l
4l
Tris-Base
30.31 g
121.24 g
Glycine
150 g
600 g
SDS
10 g
40 g
1M Tris pH 6.8
0.25 l
Tris-Base
30.29 g
Water
0.2 l
Adjust pH to 6.8, then volume. Need approximately 20 ml conc HCl !!!
1M Tris pH 8.8
0.25 l
Tris-Base
30.3 g
Water
0.2 l
Adjust pH to 8.8, then volume. Need approximately 5 ml conc HCl.
2.5M CaCl2 (Store at –20˚C)
20 ml
CaCl2, 2H2O
7.351 g
Water
15 ml
When all is dissolved, adjust volume to 20 ml, then aliquot.
2xBES (Transfection reagent)
0.2 l
2.5 l
BES, Na salt
2.352 g
29.4 g
NaCl
3.273 g
40.913 g
Na2HPO4, 2H2O (1.5 mM)
0.0534 g
0.6675 g
Note how much crystal water the Na2HPO4 has !
Adjust pH to 6.95 precisely (calibrate pH meter carefully), then volume. Check pH again.
Filter sterilize and freeze aliquots.
TE pH 7.5
0.1 l
1M Tris pH7.5
1 ml
0.5 M EDTA pH8.0
0.2 ml
Water up to
0.1 l
10x loading dye (DNA gels)
10 ml
Bromophenol blue
0.025 g
Xylene cyanol
0.025 g
Ficoll 400
2.5 g
Water up to
10 ml
Keep stirring until dissolved!
12.5M NaOH
0.1 l
NaOH pellets
50 g
Dissolve in 80 ml of water (careful !, very caustic !), allow to cool, then adjust volume to
0.1 l
10% SDS
0.1 l
SDS
10 g
Water
80 ml
After SDS is dissolved, adjust volume to 0.1 l
10x transfer buffer (Western)
0.5 l
2l
Tris-Base
15 g
60 g
Glycine
72 g
288 g
When ready to use, mix 1 volume 10x buffer, 7 volumes water, and 2 volumes methanol.
Store et 4 ˚C.
10xTBS Tween
0.5 l
1M Tris pH 7.5
0.25 l
5M NaCl
0.15 l (or 43.83 g NaCl powder)
Tween-20
2.5 ml
Adjust volume to 0.5 l after stirring.
3M NaAc, pH 5.5
0.1 l
NaAc, 3H2O
40.8 g
Water
70 ml
(Note how much crystal water the NaAc has !, e.g. it may be anhydrous)
Adjust pH with glacial acetic acid, then volume to 0.1 l
2xDB (dissociation buffer, or Laemmli buffer)
20 ml
SDS
1g
50 % glycerol
10 ml
1M Tris pH 6.8
1.25 ml
Bromophenol blue until
0.0075 %
Water up to
20 ml final volume
Aliquot into 1 ml portions and freeze. Before use, add 50 µl -mercaptoethanol to a 1 ml
aliquot.
TFB (transformation buffer, Hanahan)0.5 l
KCl
3.7 g
MnCl2, 4H2O
4.45 g
CaCl2, 2H2O
0.75 g
Hexammin cobaltchloride
0.4 g
K-MES
10 ml of 0.5 M stock pH 6.3
LB medium
Bacto tryptone
10 g
Bacto yeast extract
5g
NaCl
10 g
Adjust pH to 7.0 with NaOH
LB/ agar plates
Make LB medium as described above. Add 15g/l of Bacto agar (it will not dissolve).
Autoclave immediately! Right after autoclaving, place in 55˚C water bath. Pour plates
after it is temperature equilibrated.
LB/ampicillin agar plates
Make LB/ agar as described above. Place in 55˚C water bath. After temperature has
adjusted to 55˚C, add ampicillin from a 100mg/ml stock to a final concentration of
100µg/ml. Pour plates.
20xSSC [3 M NaCl, 0.3 M Na citrate pH 7] 2 l
NaCl
350.64 g
Na citrate, 2H2O
176.46 g
1 M Na2CO3
50 ml
Na2CO3 (anhydrous)
5.3 g
Water until
50 ml
0.1 M Na3VO4
10 ml
Na3VO4
0.183 g
Water until
10 ml
Aliquot and freeze
100 mM IPTG
10 ml
IPTG
0.238 g
Water until
10 ml
Freeze in 1 ml aliquotes
NETN buffer (GST fusions)
20 mM Tris pH 8.0
100 mM NaCl
1 mM EDTA
0.5 % NP-40
EBC extraction buffer
50 mM Tris pH 8.0
120 mM NaCl
0.5 % NP-40
Coomassie stain
0.25 % w/v Coomassie R250
10 % Acetic acid
50 % Methanol
Destain
7.5 % Acetic acid
5 % Methanol
Ponceau S stain
0.5 % Ponceau S in 5 % TCA (trichloro acetic acid)
10x PBS
NaCl
80g
KCl
2g
Na2HPO4
14.4g
KH2PO4
2.4g
Water
800 ml
Adjust pH to 7.4 then the volume to 1l. Sterilize by autoclaving
Ethidium Bromide Stock solution, 10 mg/ml
Ethidium Bromide
400 mg
Water
40 ml
Store at 4 ˚C
5x Laemmli Sample buffer
SDS
1.5g
1M Tris pH 6.8
3.75ml
Bromophenol blue
0.015g
DTT
1.16g
Glycerol
7.5ml
Mix thoroughly with stirring. Add water until 15ml total volume. Make 1ml aliquots and
store at -20˚C.
1M MgCl2
MgCl2, 6H2O
10.17g
Water
50ml final volume
0.5M NaF
NaF
2.10g
Water
100 ml final volume
100 mM PMSF (phenylmethanesulfonyl fluoride)
PMSF
0.174g
2-propanol
10 ml final volume
Store at room temperature
RIPA buffer
50 mM Tris pH 8.0
150 mM NaCl
1% NP-40
0.5% sodium deoxycholate
0.1% SDS
CSK (Cytoskeleton buffer)
10mM PIPES pH7.0
100mM NaCl
300mM Sucrose
3mM MgCl2
0.5M EGTA
19.02 g EGTA Na salt
Distilled water to 90ml
Adjust pH to 7.00 with NaOH, then volume to 100ml
1M DTT
1.54 g dithiothreitol (DTT)
33.3 µl 3M NaAc (pH 5.3)
Distilled water to 10 ml final volume. Filter sterilize and store at -20˚C in 1 ml aliquots.
0.5M LiCl/ 20 mM Tris pH 7.5
2.12 g LiCl (anhydrous)
2 ml 1 M Tris pH 7.5
Distilled water to 100 ml final volume. Store at 4˚C