A B C D
E F G H
I J K L
M N O P
Q R S T
U V W X
Y Z
A
B
Block soln for disc-staining
| wash | 5 ml | 10 ml |
| BSA | 20 mg | 40 mg |
- final [BSA] = 5mg/ml
C
Coomassie Brilliant Blue stain
| Coomassie Brilliant Blue R250 | 0.25g |
| MetOH:H2O (1:1 v/v) | 90ml |
| Glacial acetic acid | 10ml |
- filter the solution through a Whatman No. 1 filter to remove any particulate matter
- Destain for Coomassie gel is the same as above but without the dye
D
Destain for Coomassie gel
— see Coomassie Brilliant Blue stain
E
F
Fix solution
| 0.5 M Pipes | 2 ml |
| 0.5 M EGTA | 20 µl |
| 1.0 M MgSO4 | 20 µl |
| Triton X-100 | 100 µl |
| add dH2O to | 10 ml |
- store @ 4o C for up to a week
- working soln: 950µl Fix + 50µl formeldehyde
10% Formaldehyde
| ddH2O | 70-80 ml |
| Paraformaldehyde | 10 g |
| NaOH | 1 pellet |
- stir for 30min-1hr until soln is clear (if it remains cloudy use newer formaldehyde)
- pH to 7.2-7.4 using conc HCl
- bring up to 100 ml - store @ 4o C for up to a month
G
Grinding Buffer
| 100 ml | [Final] | |
| Tris-Cl pH 8.0 | 5ml of 1M | 50 mM |
| EDTA | 10ml of 0.5M | 50 mM |
| NaCl | 2ml of 5M | 100 mM |
| SDS | 10ml of 10% | 1% |
| Bring up to: | 100ml |
H
I
J
K
L
Lysis buffer
for miniprep
| Glucose | 50 mM | |
| Tris-Cl (pH 8.0) | 25 mM | |
| EDTA (pH 8.0) | 10 mM |
- autoclave and store at 4C
M
N
NP-40 buffer
| add: | [Final] | |
| NaCl | 1.35 ml of 5M | 150mM |
| NP-40 | 450 µl | 1.0% |
| Tris | 2.25 ml of 1M | 50mM (pH 8.0) |
| add ddH2O to: | 45 ml |
- add protease inhibitors before use
O
O/N Mounting soln for disc-staining
| 1.0 M Tris (pH 8.8) | 2.5 ml |
| Glycerol | 5 ml |
| add dH2O to | 50 ml |
P
PBT
1X PBS + 0.1% Triton X-100
PBT+N
4 mls PBT
200 ul normal goat serum
PDA mounting soln for disc-stainin
stock soln:
| 30 mg PDA |
| 4 ml ddH2O |
| 6 ml glycerol |
- add glycerol last b/c it impeds disolving
- store @ -20° C covered w/ foil
working soln:
| 1 ml stock |
| 1 ml 0.5 M Tris pH 8.8 |
| 4.0 ml ddH2O |
- add liquid to 15ml conical tube wrapped in foil
PEM
| 100ml | 250ml | |
| 0.1 M Pipes | 20 ml 0.5M Pipes | 50 ml 0.5M Pipes |
| 2 mM EGTA | 400 ul 0.5 M EGTA | 1 ml 0.5 M EGTA |
| 1 mM MgSO4 | 100 ul 1 M MgSO4 | 250 ul 1 M MgSO4 |
- pH to 6.95, filter sterilize and store at 4°C
0.5 M Pipes
15.12g in 100 ml. pH to 6.95 and filter sterilize
4M KoAC pH 5.5
| 100ml | |
| KoAc | 39.26 |
Renew Buffer (for Qiagen tips)
| NaCl | 3M |
| Triton X-100 | 0.15% |
RIPA Buffer
| NaCl | 150mM | 1.35 ml of 5M |
| NP-40 | 1% | 450 µl |
| SDS | 0.1% | 450 µl 10% SDS |
| Tris | 50mM | 2.25 ml of 1M pH 7.5 |
| DOC | 0.5% |
- add ddH2O to 45 ml
- add protease inhibitors before use
5x Running Buffer
for SDS-PAGE gels
| Tris base | 60.5g | 121g |
| Glycine | 288g | 576g |
| EDTA | 7.44g | 14.88g |
| SDS | 10g | 20g |
| adjust volume to: | 2L | 4L |
SDS-PAGE Gels
|
5% |
7.50% |
8.75% |
10% |
12.50% |
15% |
|
|
H2O |
5.8 ml |
5 ml |
4.5 ml |
4.2 ml |
3.3 ml |
2.5 ml |
|
Tris buffer |
2.5 ml |
2.5 ml |
2.5 ml |
2.5 ml |
2.5 ml |
2.5 ml |
|
Acrylamide |
1.7 ml |
2.5 ml |
2.9 ml |
3.3 ml |
4.2 ml |
5 ml |
|
15% APS |
30 µl |
30 µl |
30 µl |
30 µl |
30 µl |
30 µl |
|
TEMED |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
SDS-PAGE Lower Gel Buffer (4x)
| Tris base | 36.34g |
| 10% SDS | 8ml |
| ddH2O | ~170ml |
| (1:1) HCl acid | till pH 8.8 |
| adjust volume to: | 200ml |
SDS-PAGE Upper Gel Buffer (4x)
| Tris base | 12.12g |
| 10% SDS | 8ml |
| ddH2O | ~170ml |
| (1:1) HCl acid | ~5 to 6 mls till pH 6.8 |
| adjust volume to: | 200ml |
| [Final] | ||
| 1M Tris-Cl (pH 6.8) | 2.5ml | 250mM |
| SDS | 0.82g | 8.2% |
| Bromphenol Blue | 5mg | 0.05% |
| BME | 0.5ml | 5% |
| Glycerol | 4ml | 40% |
| adjust volume to: | 10ml |
Solution I see Lysis
buffer
Solution II
| [Final] | ||||||
| NaOH | 0.12 g | 0.11 | 0.10 | 0.09 | 0.8 g | 0.2M |
| SDS | 1.5 ml of 10% | 1.38 | 1.25 | 1.13 | 1 ml of 10% | 1% |
| Bring up to 15ml | 13.8 | 12.5 | 11.3 | Bring up to 10ml |
| Potassium acetate | 44.07g in 60ml ddH2O |
| Glacial acetic acid | 11.5 ml |
| ddH2O | 28.5 ml |
- the resulting solution is 3M potassium and 5M acetate
Stripping Buffer for Western Blot
| add: | [Final] | |
| Tris pH 7.0 | 31.25ml of 1.0M Tris pH7.0 | 62.5mM |
| ß-ME | 3.41ml | 100mM |
| SDS | 100ml of 10%SDS | 2% |
| adjust volume to: | 500ml |
- To strip blot incubate at 50° C for 30 min with occasional shacking
| Tris base | 11.6g | 23.2g | 34.8g | 46.4g |
| Glycine | 58g | 116g | 174g | 232g |
| SDS | 2g | 4g | 6g | 8g |
| MetOH | 400ml | 800ml | 1.2L | 1.6L |
| adjust volume to: | 2L | 4L | 6L | 8L |
| 1x | 5x | |
| 1M Tris-Cl (pH 7.5) | 100ml | 500ml |
| ddH2O | 900ml | 495ml |
| NaCl | 9g | 45g |
| Tween-20 | 1ml | 5ml |
U
V
W
Wash soln for disc-staining
| 1.0 M Tris (pH 6.8) | 10 ml | 5 ml |
| 5.0 M NaCl | 6 ml | 3 ml |
| add H2O to | 200 ml | 100 ml |
Then add:
| BSA | 0.2 g | 0.10 g |
| NP-40 | 1 ml | 0.5 ml |
- stir until dissolved - store @ 4o C for up to a week
X
Y
Z