Griffitts:Common buffers

2% bromophenol blue
Sterile filter
 * 0.4 g bromophenol blue (BPB)
 * 20 mL 50 mM Tris pH 8.0

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
Note: use 5 μL per lane; 3kb band = 60 ng
 * 147 μL ddH20
 * 34 μL 8X DNA loading buffer
 * 20 μL 1 kb ladder from NEB
 * 2 μL 0.25 M EDTA

Protein ladder
Note: use 5 μL per lane
 * 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

ethidium bromide (10 mL)
Note: use 8 μL per 50 mL gel
 * 10 mL ddH20
 * 10 mg ethidium bromide

50X TAE (500 mL)
Stir until EDTA is dissolved. Adjust final pH to 8.0 to 8.1 using NaOH pellets. Note: this is half the amount of EDTA compared to standard TAE.
 * 400 mL ddH20
 * 121 g Trizma base
 * 28 mL glacial acetic acid
 * 4 g Na2EDTA

10X Laemmli running buffer (1 L)
QS to volume with ddH20
 * 30.3 g Trizma base (Tris)
 * 144.2 g glycine
 * 10 g sodium dodecyl sulfate (SDS)

Coomassie stain (300 mL)
Mix for 30 min.
 * 120 mL ethanol
 * 0.3 g Brilliant Blue R
 * 30 mL acetic acid
 * 150 mL ddH20

Coomassie destain solution (1 L)

 * 550 mL ddH20
 * 400 mL ethanol
 * 50 mL acetic acid

colony lysis solution (for PCR) (100 mL)
Note: Store in fridge
 * 96.2 mL of sterile ddH2O
 * 0.5 mL of 1 M Tris pH 8.0 (final concentration: 5 mM)
 * 0.8 mL of .25 M EDTA (final concentration: 2 mM)
 * 2.5 mL of 20% Triton X-100 (final concentration: 0.5%)
 * in fridge with the antibiotics

TE buffer
For 75 mL T3E0.3: Note: T3E0.3 is what we use most of the time in the lab For 75 mL T10E1: Note: T10E1 is what we use for genomic DNA preps For 75 mL T50E10: Note: T50E10 is what we use for DNA midi-preps
 * 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
 * 74.37 mL ddH2O (i.e. remove 630 μL from 75 mL ddH2O)
 * 450 μL 1 M Tris pH 8.0
 * 180 μL .25 M EDTA
 * 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)
Autoclave
 * 75 mL ddH2O
 * 9.09 g Tris (Trizma base)
 * pH to 6.8 with HCl

1 M Tris, pH 7.5 (75 mL)
Autoclave
 * 75 mL ddH2O
 * 9.09 g Tris (Trizma base)
 * pH to 7.5 with HCl

1 M Tris, pH 8.0 (75 mL)
Autoclave
 * 75 mL ddH2O
 * 9.09 g Tris (Trizma)
 * pH to 8.0 with HCl

1 M Tris, pH 8.8 (75 mL)
Autoclave
 * 75 mL ddH2O
 * 9.09 g Tris (Trizma)
 * pH to 8.8 with HCl

0.25 M EDTA (75 mL)
Autoclave
 * 75 mL ddH2O
 * 6.98 g EDTA
 * pH to 8.0 with NaOH

10% Triton X-100 (40 mL)
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly Note: do not filter sterilize Store at 4°C
 * 36 mL dH2O
 * 4 mL Triton X-100

20% Triton X-100 (40 mL)
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly Note: do not filter sterilize Store at 4°C
 * 32 mL dH2O
 * 8 mL Triton X-100

0.5 M MOPS (40 mL)
QS to volume with ddH2O
 * 20 mL ddH2O
 * 4.19 g MOPS
 * pH to 7.0 (~3.5 mL NaOH)

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
Note: RNAse A is purchased from Sigma (R-6513)
 * 5 mg/mL RNAse A (in freezer)
 * 10 mM Tris pH 8.0

silica suspension
Store at -20°C
 * Wash 5X with ddH20
 * Resuspend as 50% slurry in ddH20

silica wash solution

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

Proteinase K
Store at -20°C
 * 20 mg/mL Proteinase K (in freezer)
 * 20 mM Tris pH 8.0

Lysozyme
Store at -20°C
 * 1 mL dH2O
 * 30mg lysozyme (in freezer)
 * Dilute 60X into lysis buffer

2N KOH (40 mL)
NOTE: for KOH 2N = 2M
 * 4.49 g KOH pellets
 * 40 mL ddH2O

2N NaOH (40 mL)
NOTE: for NaOH 2N = 2M
 * 3.2 g NaOH pellets
 * 40 mL ddH2O