bottle wrapped with aluminum foil. Avoid skin contact with
unpolymerized acrylamide.
2. Ammonium persulfate: 10% solution in water.
3. Sodium dodecyl sulfate (SDS): 10% solution in water.
4. N,N,N,N-Tetramethylethylenediamine (TEMED): store at
4
C.
5. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Transfer
181.7 g Tris to a graduated cylinder or a glass beaker and
then add about 900 mL of water and mix. Adjust the pH
with HCl and add more water to a final volume of 1 L. Autoclave the solution (121
C, 20 min) and store at room
temperature.
6. Stacking gel buffer: 1 M Tris–HCl, pH 6.8. Dissolve 121.14 g
Tris in 900 mL water, adjust pH to 6.8 with HCl, and then
complete the volume to 1 L. Autoclave and store at room
temperature.
7. SDS-PAGE sample buffer (5Â): 250 mM Tris–HCl, pH 6.8,
0.1% SDS, 50% glycerol, 6.7% β-mercaptoethanol, 0.5 mg/mL
bromophenol blue. Mix 7.5 mL of 1 M Tris–HCl, pH 8.0,
3 mL of 10% SDS, 15 mL of glycerol, and 15 mg of bromophenol blue, and complete the volume to 28 mL. Heat the
solution at 65
C until the dye is completely dissolved, and then
add 2 mL β-mercaptoethanol.
8. Tris-glycine buffer (5Â): 125 mM Tris–HCl, pH 8.3, 1.25 M
glycine, 0.5% SDS. Keep the solution at room temperature.
9. Glass plates, gel caster, and combs.
10. Nitrocellulose membranes.
11. Transfer buffer (5Â): Mix 625 mL of 5Â Tris-glycine buffer,
18.5 mL of 10% SDS, 356.5 mL of water.
12. Phosphate buffered saline (PBS, 10Â): 1.37 M NaCl, 27 mM
KCl, 100 mM Na 2 HPO 4 , 20 mM KH 2 PO 4 , pH 7.4. Autoclave
and store at room temperature.
13. PBS-Tween 20 (PBS-T): 1Â PBS, 0.05% Tween 20. Since this
detergent is viscous, pipetting should be done slowly.
14. Blocking buffer: PBS-T, 10% dry milk, or 5% bovine serum
albumin (BSA). Weigh out 10 g of skimmed dry milk or 5 g of
BSA and dissolve the powder in 100 mL of PBS-T.
15. Plastic container.
16. Ponceau S.
17. Semidry transfer system.
18. Filter paper.
Gene Silencing by Single-Guide RNA and dCas9
113
unpolymerized acrylamide.
2. Ammonium persulfate: 10% solution in water.
3. Sodium dodecyl sulfate (SDS): 10% solution in water.
4. N,N,N,N-Tetramethylethylenediamine (TEMED): store at
4
C.
5. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Transfer
181.7 g Tris to a graduated cylinder or a glass beaker and
then add about 900 mL of water and mix. Adjust the pH
with HCl and add more water to a final volume of 1 L. Autoclave the solution (121
C, 20 min) and store at room
temperature.
6. Stacking gel buffer: 1 M Tris–HCl, pH 6.8. Dissolve 121.14 g
Tris in 900 mL water, adjust pH to 6.8 with HCl, and then
complete the volume to 1 L. Autoclave and store at room
temperature.
7. SDS-PAGE sample buffer (5Â): 250 mM Tris–HCl, pH 6.8,
0.1% SDS, 50% glycerol, 6.7% β-mercaptoethanol, 0.5 mg/mL
bromophenol blue. Mix 7.5 mL of 1 M Tris–HCl, pH 8.0,
3 mL of 10% SDS, 15 mL of glycerol, and 15 mg of bromophenol blue, and complete the volume to 28 mL. Heat the
solution at 65
C until the dye is completely dissolved, and then
add 2 mL β-mercaptoethanol.
8. Tris-glycine buffer (5Â): 125 mM Tris–HCl, pH 8.3, 1.25 M
glycine, 0.5% SDS. Keep the solution at room temperature.
9. Glass plates, gel caster, and combs.
10. Nitrocellulose membranes.
11. Transfer buffer (5Â): Mix 625 mL of 5Â Tris-glycine buffer,
18.5 mL of 10% SDS, 356.5 mL of water.
12. Phosphate buffered saline (PBS, 10Â): 1.37 M NaCl, 27 mM
KCl, 100 mM Na 2 HPO 4 , 20 mM KH 2 PO 4 , pH 7.4. Autoclave
and store at room temperature.
13. PBS-Tween 20 (PBS-T): 1Â PBS, 0.05% Tween 20. Since this
detergent is viscous, pipetting should be done slowly.
14. Blocking buffer: PBS-T, 10% dry milk, or 5% bovine serum
albumin (BSA). Weigh out 10 g of skimmed dry milk or 5 g of
BSA and dissolve the powder in 100 mL of PBS-T.
15. Plastic container.
16. Ponceau S.
17. Semidry transfer system.
18. Filter paper.
Gene Silencing by Single-Guide RNA and dCas9
113