Buffer Prep
1 M Tris-HCl Buffers
| pH | Volume (L) | TrisBase(g) | HCl (ml) |
|---|---|---|---|
| 7.0 | 2 | 242.2 | 150-155 |
| 7.5 | 2 | 242.2 | 120-125 |
| 8.0 | 2 | 242.2 | 80-85 |
Autoclavable.
EDTA 0.5 M (pH8.0)
0.5M, 1L: 148 g EDTA
+ ~30-40 g NaOH to adjust pH
(or 186 g EDTA-Na.2H2O + ~20 g NaOH)
Note: pH adjusted by NaOH is essential for solubility.
Autoclavable.
TAE DNA Electrophoresis Buffer(50 X)
(2 M Tris, 50 mM EDTA) 2 L 484 g Tris 114.2 ml glacial acetic acid 200 ml 0.5 M EDTA 8.0 To make 1x TAE 20 L, add 400 ml 50X buffer into 19.6 L ddH2O.
SDS-PAGE Gel Solutions
| Solution | Vol (L) | Tris (g) | HCl (ml) | 10% SDS (ml) |
|---|---|---|---|---|
| 4x Lower gel buffer 1.5 M Tris-Cl, pH 8.8, 0.4% SDS | 2 | 363.3 | 50-60 | 80 ml |
| 4x Upper gel buffer 0.5 M Tris-Cl, pH 6.8, 0.4% SDS | 2 | 121.1 | 70-80 | 80 ml |
10% SDS
1L: 100g SDS into 1 L, heat to 68 degrees Celsius for solubility. pH ~6.6.
5X SDS Loading Sample Buffer
| Reagent | Stock solution | Add volume |
|---|---|---|
| 250 mM TrisHCl pH6.8 | 2 M | 12.5 ml |
| 10% SDS | 100% | 10 g |
| 30% Glycerol | 100% | 30 ml |
| 5% β-mercapitalethanol (or 0.5M DTT) | 100% | 5 ml |
| 0.02% bromophenol blue | 0.04% | 52 ml |
6X DNA loading sample buffer
(40% sucrose, 0.01-0.02% BPB)
100 ml
Add 40 g sucrose to 50 ml 0.04% BPB solution, adjust final volume 100 ml.
SDS-PAGE Electrophoresis Running Buffer (10x)
(1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3)
10 L.
303 g Trisbase (FW 121.1)
1440 g glycine (FW 75.07)
100 g SDS
No need to adjust pH
Transfer Buffer without SDS (10x)
(1x: 25 mM Tris, 192 mM glycine, pH8.3)
10 L
303 g Trisbase,
1440 g glycine
No need to adjust pH
Transfer Buffer (1x)
500 ml
50 ml of 10x Transfer buffer (without SDS) or 10x SDS-PAGE running buffer (w/ SDS)
100 ml of Methanol (final 20% methanol)
350 ml ddH2O
TBS (10x)
(1x: 150 mM NaCl, 10 mM Tris pH8.0)
10 L
876.6 g NaCl (FW 58.44),
121.1 g Tris,
~40 ml HCl
to pH8.0
TBS-T (1x)
1L
100 ml 10x TBS
10 ml 10% Tween20 (final 0.1% v/v)
890 ml ddH2O
Block buffer
(5% Nonfat milk in TBS-T)
5g milk in 100 ml TBST
NaCl 4 M
2 L: 467.5 g NaCl.
Autoclavable
NaOH 10 M
0.5 L: 200 g
NaAc 3 M
500 ml: add 204 g NaAc.3H2O (FW 136), adjust pH by glacial acetic acid (~60 ml) to pH5.2.
Autoclavable.
MgCl2 1M
500 ml: Add 101.65 g MgCl2.6H2O into 500 ml ddH2O.
Autoclavable.
CaCl2 1M
400 ml: Add 58.8 g CaCl2.2H2O (FW 147), filter for sterilization.
Dilute 10x to make 100 mM CaCl2.
MgSO4 1M
500 ml: Add 123.3 g MgSO4.7H2O into 500 ml ddH2O.
Autoclavable.
ZnCl 0.5M
50 ml: 3.4 g ZnCl to 50 ml.
