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Ampicillin (1000X,
100mg/ml)
1.0 g
Ampicillin
Add dH2O
to 10 ml
Sterile
filter with 0.2um filter
Aliquot
~ 1ml, freeze @ -20°C
IPTG (1000X, 1M)
2.382 g
IPTG
Add dH2O
to 10 ml
Sterile
filter with 0.2um filter
Aliquot
~ 1ml, freeze @ -20°C
Kanamycin (1000X,
50mg/ml)
682 mg kanamycin sulfate
(Invitrogen stock pwd is 0.734 ug/mg pwd)
Add dH2O
to 10 ml
Sterile
filter with 0.2um filter
Aliquot
~ 1ml, freeze @ -20°C
G418 (100mg/ml stock)- use 1mg/ml final
In hood,
1.4 g G418 (di)sulfate powder
(based on active weight, usually ~700ug active/mg powder)
10 ml sterile dH2O
Sterile filter with 0.2um
filter into sterile tube
Use 1 mg/ml in media for
selection
100 ul per 6 ml
of media gives ~1.7mg/ml
Refresh with new media/G418 every 3 days
LB Agar
16.0 g
LB
7.5 g
Agar
500 ml
dH2O
Autoclave, cool
Add
antibiotic, pour
Store
at 4°C
PBS (10X)
(FINAL CONC IN PARENTHESIS)
80.0 g NaCl (1.37
M)
2.0 g KCl
(26.8 mM)
14.2 g Na2HPO4
or 27.0 g Na2HPO4*7H2O
(0.1 M)
2.4 g KH2PO4
(17.6 mM)
dH2O
to 800 ml stirring in beaker
Adjust pH to 7.4
Adjust to 1 L
Autoclave
10X SDS-PAGE Running
Buffer-Denaturing
29 g
Tris Base
144 g
Glycine
10 g
SDS or 50 ml of a 20% SDS solution
dH2O
to 1L
Do NOT
adjust pH
For Non-Denaturing,
same as above w/out SDS
2X SDS-Sample Buffer
25 ml
0.5M Tris-HCL, pH 6.8
20 ml
glycerol
20 ml
20% SDS
5 ml
0.1% BPhB
dH2O
to 100 mL
Agarose Gel Separation
Range
Agarose %
Range of Separation
0.3
60 - 5 kb
0.6
20 - 1 kb
0.7
10 - 0.8 kb
0.9
7 - 0.5 kb
1.2
6 - 0.4 kb
1.5
4 - 0.2 kb
2.0
3 - 0.1 kb
Glycerol Bacterial
Stock
1500 ul bacterial broth
500 ul sterile 60%
glycerol
Label, freeze at -80°C
TBST (10X)
30 g Tris Base
88 g NaCl
2 g KCl
970 mL dH2O
Bring pH to 7.5
5 ml Tween-20
Adjust to 1 L
TBS (10X)
30 g Tris Base
88 g NaCl
2 g KCl
970 mL dH2O
Bring pH to 7.5
Adjust to 1 L
Immunoglobulin
Stripping Buffer & Protocol
0.8 ml
b-ME
10 ml of 20% SDS
12.5 ml 0.5 M Tris-HCl pH6.8
qs with dH2O to 100 mL
Protocol
1) Add membranes
and buffer to small plastic box with tight fitting lid
2) Incubate 50°C
for 45 minutes
3) Rinse membrane
with dH2O
4) Wash 3x in TBST,
5 min each
5) Take on to
blocking step
Glycine Stripping
Dissolve 188mg glycine in
75 ml dH2O
Adjust pH to 2 with HCl
Add 5ml 20% SDS (1%
final)
QS to 100 ml with dH2O
Protocol
1) Warm buffer to
50°C for 15-20 min
2) Soak blot in
buffer in container with lid at RT for 30 min
3) Rinse blot with
dH2O 4-5 times
4) Wash 3 times
with TBST ~5 min each
5) Take on to
blocking step
Wet Immunoblot Transfer
Buffer (10X)
30.25 g Tris Base
144 g Glycine
Add 0.1% SDS only if
protein of interest >100KDa
For 1X, dilute to by:
100 ml 10X Transfer
Buffer
200 ml MeOH
700 ml dH2O
Semi-Dry Immunoblot
Transfer Buffer (1X)
5.82 g Tris Base
(48mM)
2.93 g Glycine
(39mM)
Dissolve in 700 ml dH2O
Add 200 ml MeOH
Add 3.75 ml of 10% SDS
(or 1.875 ml of 20% or 0.0375 g SDS) (0.0375%)
Adjust to 1 L
pH should be ~9.2, do NOT
adjust
TAE (10X)
48.4 g Tris Base
11.42 ml Glacial Acetic
Acid
20 ml 0.5 M EDTA (pH 8)
dH2O to 1 L
RIPA
Buffer (1X) (Denaturing)
25 mM Tris-HCl (pH 7.6)
150 mM
NaCl
1% NP40
1% Na
Deoxycholate
0.1% SDS
-
Add 12.5 ml of 1 M Tris-HCl
-
Add 15 ml of 5 M NaCl
-
Add 5 ml of NP40
-
Add 50 ml of 10% NaDeoxycholate stock, or add 5 g of NaDeoxycholate
powder
-
Add 2.5 ml of 20% SDS or add 0.5 g of SDS powder
- dH2O to
500 ml
For
non-denaturing (e.g., Co-IP), remove SDS and Na Deoxycholate,
replace NP40 above with 0.5% final concentration.
Krebs-Ringer Bicarbonate Buffer
1.5mM MgSO4
4.7mM KCl
1.3 mM CaCl2
1.2mM KH2PO4
25mM NaHCO3
11.7mM Glucose
118mM NaCl
pH Adjusted on day of use with O2/CO2 Gas
- 369.7mgMgSO4
-350 mg KCl
-191mg CaCl2
-163.3mg KH2PO4
-2.1g NaHCO3
-2.108g Glucose
-6.895g NaCl
-1000ml diH2O
HENS
25mM HEPES pH 7.5
1mM EDTA
1% SDS
10µM Neocuproine
-Add 3.25g of HEPES
-186mg EDTA
-1mg Neocuproine
-5g SDS
-450 ml diH2O
-Adjust pH to 7.5
-qs to 500ml
For HENS10 dilute HENS buffer 1:10 in diH2O
Neutralization Buffer (for HENS protocol)
20mM HEPES pH 7.5
1mM EDTA
100mM NaCl
0.5% Triton
-Add 2.6g HEPES
-186mg EDTA
-2.922g NaCl
-450ml diH2O
-Adjust pH to 7.5
-Add 2.5 ml Triton-X 100
-qs to 500ml
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