Buffer Prep

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GroseLab:Protocols

Contents

1 M Tris-HCl Buffers

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

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

100 ml
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.

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