Afterward, beads were washed twice with lysis buffer, and 40 [micro]L of 2x sample buffer (100 mmol/L Tris, pH 6.80; 40 g/L SDS; 20 mL/L glycerol; 20 /[micro]g/L bromphenol blue
; 15 g/L dithiothreitol) was added to each tube.
We prepared samples for electrophoresis by adding 20 [micro]L of sample buffer (40 mmol/L Tris, 33 g/L SDS, 500 mL/L glycerol, and bromphenol blue
) to 80 [micro]L of eluate.
The amplicons were mixed with equal amounts of single-strand conformation polymorphism (SSCP) buffer containing 950 mL/L formamide, 10 mmol/L NaOH, 2.5 g/L bromphenol blue
, and 2.5 g/L xylene cyanol and then submitted to mutation screening by SSCP and heteroduplex analysis.
We then added 110 [micro]L of rehydration solution (7 mol/L urea, 2 mol/L thiourea, 40 g/L CHAPS, 60 mmol/L DTT, 5 mL/L IPG buffer pH 3-10 NL, and a trace of bromphenol blue
) to the above mixture.
Gelatinase calibrators were prepared by diluting healthy capillary blood with 15 volumes of nonreducing sample buffer (containing, per liter, 62.5 mmol of Tris-HCl, pH 6.8; 350 mL of glycerol; 25 mL of Triton X-100; 40 g of sodium dodecyl sulfate; 0.2 g of Brij-35; 0.02 g of Na[[N.SUB.3]; and 0.1 g of bromphenol blue
We denatured 20 [micro]L of sample, containing 5-50 ng of protein, by heating in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer [50 mmol/L Tris-HCl (pH 6.8), 0.1 mmol/L dithiothreitol, 20 g/L SDS, 1 g/L bromphenol blue
, 100 g/L glycerol] at 75[degrees]C for 10 min and run on a 4-12% polyacrylamide/ SDS gel (Invitrogen).
We diluted 1 [micro]L of human EDTA plasma that had been stored for up to 2 years at -70 [degrees]C in 20 [micro]L of sample loading buffer [10 g/L sodium dodecyl sulfate, 100 mL/L glycerol, 25 mmol/L Tris (pH 6.8), 0.05 g/L bromphenol blue
, and 50 mL/L ([beta]-mercaptoethanol] and boiled the mixture for 5 min.
For SSCP and heteroduplexes analysis, PCR products were mixed with equal amounts of SSCP buffer containing 950 mL/L formamide, 10 mmol/L NaOH, 2.5 g/L bromphenol blue
, and 2.5 g/L xylene cyanol.
A 2-[micro]L portion of the PCR product was diluted with 4 [micro]L of the SSCP buffer (475 mL/L formamide, 0.5 g/L xylene cyanol, 0.2 g/L bromphenol blue
, 5 mmol/L Tris, and 0.5 mmol/L EDTA).
Ten microliters each of the PCR product and the loading buffer (980 mL/L formamide, 0.1 g/L bromphenol blue
, 0.1 g/L xylene cyanol, and 10 mmol/L EDTA) were mixed and boiled in water for 5 min.