Common Buffers

Electrophoresis Buffers

2% bromophenol blue

0.4 g bromophenol blue (BPB)
20 mL 50 mM Tris pH 8.0

Sterile filter

8X DNA loading

40% glycerol
1/50 dilution of 2% bromophenol blue

6X SDS loading

7.5 mL 80% glycerol
2.5 mL 1 M Tris pH 6.8
1.2 g SDS
600 μL 2-mercaptoethanol (in fume hood)
150 μL 2% bromophenol blue

6X NAT loading

7.5 mL 80% glycerol
2.5 mL 1 M Tris pH 6.8
5 μL 2-mercaptoethanol (in fume hood)
150 μL 2% bromophenol blue

10X NAT running

3 g Tris Base
14.4 g glycine
dH₂O to 100 mL

DNA ladder

147 μL ddH₂0
34 μL 8X DNA loading buffer
20 μL 1 kb ladder from NEB
2 μL 0.25 M EDTA

Note: use 5 μL per lane; 3kb band = 60 ng

Protein ladder

Open vial of Sigma SDS6H2-1VL
Add 1 mL ddH₂0
Add 500 μL 6X SDS loading
Aliquot 50 μL per tube and store at -20°C

Note: use 5 μL per lane

ethidium bromide (10 mL)

10 mL ddH₂0
10 mg ethidium bromide

Note: use 8 μL per 50 mL gel

50X TAE (500 mL)

400 mL ddH₂0
121 g Trizma base
28 mL glacial acetic acid
4 g Na₂EDTA

Stir until EDTA is dissolved.
Final pH should be between 8.1 and 8.4
Note: this is half the amount of EDTA compared to standard TAE.

10X Laemmli running buffer (1 L)

30.3 g Trizma base (Tris)
144.2 g glycine
10 g sodium dodecyl sulfate (SDS)

QS to volume with ddH₂0

Coomassie stain (300 mL)

120 mL ethanol
0.3 g Brilliant Blue R

Mix for 30 min.

30 mL acetic acid
150 mL ddH₂0

Coomassie destain solution (1 L)

550 mL ddH₂0
400 mL ethanol
50 mL acetic acid

DNA Extraction Buffers

colony lysis solution (for PCR) (75 mL)

72.15 mL of sterile ddH₂O (i.e. remove 2.85 mL from a 75-mL bottle of sterile ddH₂O)
375 μL of 1 M Tris pH 8.0 (final concentration: 5 mM)
600 μL of .25 M EDTA (final concentration: 2 mM)
1.875 mL of 20% Triton X-100 (final concentration: 0.5%)
- in fridge with the antibiotics

Note: Store in fridge

TE buffer

For 75 mL T₃E_0.3:

74.685 mL ddH₂O (i.e. remove 315 μL from 75 mL ddH₂O)
225 μL 1 M Tris pH 8.0
90 μL .25 M EDTA

Note: T₃E_0.3 is what we use most of the time in the lab

For 75 mL T₅E_0.5:

74.475 mL ddH₂O (i.e. remove 525 μL from 75 mL ddH₂O)
375 μL 1 M Tris pH 8.0
150 μL .25 M EDTA

For 75 mL T₁₀E₁:

73.95 mL ddH₂O (i.e. remove 1.05 mL from 75 mL ddH₂O)
750 μL 1 M Tris pH 8.0
300 μL .25 M EDTA

For 75 mL T₅₀E₁₀:

68.25 mL ddH₂O (i.e. remove 6.75 mL from 75 mL ddH₂O)
3.75 mL 1 M Tris pH 8.0
3 mL .25 M EDTA

1 M Tris, pH 6.8 (75 mL)

75 mL ddH₂O
9.09 g Tris (Trizma base)
pH to 6.8 with HCl

Autoclave

1 M Tris, pH 7.5 (75 mL)

75 mL ddH₂O
9.09 g Tris (Trizma base)
pH to 7.5 with HCl

Autoclave

1 M Tris, pH 8.0 (75 mL)

75 mL ddH₂O
9.09 g Tris (Trizma)
pH to 8.0 with HCl

Autoclave

1 M Tris, pH 8.8 (75 mL)

75 mL ddH₂O
9.09 g Tris (Trizma)
pH to 8.8 with HCl

Autoclave

0.25 M EDTA (75 mL)

75 mL ddH₂O
6.98 g EDTA
pH to 8.0 with NaOH

Autoclave

10% Triton X-100 (40 mL)

36 mL dH₂O
4 mL Triton X-100

Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C

20% Triton X-100 (40 mL)

32 mL dH₂O
8 mL Triton X-100

Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C

0.5 M MOPS (40 mL)

20 mL ddH₂O
4.19 g MOPS
pH to 7.0 (~3.5 mL NaOH)

QS to volume with ddH₂O

Plasmid Purification Solutions

Resuspension Buffer

68.25 mL ddH₂O (i.e. remove 6.75 mL from 75 mL ddH₂O)
3.75 mL 1 M Tris pH 8.0
3 mL .25 M EDTA

lysis solution

1% SDS
200 mM NaOH

Neutralization solution

4 M potassium acetate
pH to 5.5 with acetic acid

RNAse A

20 mg/mL RNAse A (in freezer)
10 mM Tris pH 8.0

Note: RNAse A is purchased from Sigma (R-6513)

silica suspension

Wash 5X with ddH₂0
Resuspend as 50% slurry in ddH₂0

Store at -20°C

silica wash solution

50% ethanol
10 mM Tris pH 7.5
50 mM NaCl
2.5 mM EDTA

Proteinase K

20 mg/mL Proteinase K (in freezer)
20 mM Tris pH 8.0

Store at -20°C

Lysozyme

1 mL dH₂O
100mg lysozyme (in freezer)

Store at -20°C

Bases

2N KOH (40 mL)

4.49 g KOH pellets
40 mL ddH₂O

NOTE: for KOH 2N = 2M

2N NaOH (40 mL)

3.2 g NaOH pellets
40 mL ddH₂O

NOTE: for NaOH 2N = 2M