DNA Content Assay.doc

2020/01/19DNA定量准备：黑色96-well plate costar 391515 mL离心管 1个10 mL移液管 1个Multi-Eppendof Pipette1.25mL 自动Multi-pipette adapter2M NaCl 25mM Tris 15mL 保存于低温室Hochest 33258 -20分装保存（10mg/ml） DNA标准品Stock solution -20分装保存（1mg/ml,20L/tube） 步骤：1. 室温下溶解DNA标准品及样本； 2. 开启plate reader电源，打开Ascent software，设定程序；3. DNA标准品用2M NaCl 25mM Tris稀释：No.S1S2S3S4S5S6Conc. (g/mL)0.5124816（stock）Stock solution的制备：1mL TE(PH=8.0)溶解1mgDNA（Sigma PD 3664）=1mg/ml DNA 标准品Stock solution。 然后分装20L/tube备用。Room#309 -20 山下取16L stock solution (1mg/ml)+984L 2M NaCl 25mM Tris=1000L 16g/mL;- 500L取500L 16g/mL+500L 2M NaCl 25mM Tris=1000L 8g/mL; - 500L取500L 8g/mL+500L 2M NaCl 25mM Tris=1000L 4g/mL; - 500L取500L 4g/mL+500L 2M NaCl 25mM Tris=1000L 2g/mL; - 500L取500L 2g/mL+500L 2M NaCl 25mM Tris=1000L 1g/mL; - 500L取500L 1g/mL+500L 2M NaCl 25mM Tris=1000L 0.5g/mL; - 1000LSIGMA PD3664-1MG（低温室）(vial)，TE（pH=8.0）（open lab）1mL入溶、20L分注。(dispensing in 20L )4. 移取10mL 2M NaCl 25mM Tris入15mL离心管中，再加入5L H33258，用锡箔纸遮光置于冰上；5. 96孔板中每孔加入样本或标准品中100L，空白对照中加入100L 2M NaCl 25mM Tris；6. 用1.25mL自动加样器每孔加入25L H33258（即步骤4配制的溶液）；7. Spin down 5sec, 遮光置于摇床上混匀10分钟；8. 置于plate reader中，356nm波长处激活，458nm波长测量并保存数据；9. 数据保存为：日期.xls。10. Thermo Fluoroskan Ascent;11. Click “Ascent software”;12. Open: ASCSSW26-AscData/hou1-SAWADA1. SEF-OK-Plate out-Plate in-Start；13. Copy data area-open a new.xls-paste data-saved as 123.xls-saved desk-Hou.file.DNA Content Assay:Standards: 6; Blank:1; Samples:17123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6X1X1X1CX2X2X2X3X3X3X4X4X4X5X5X5DX6X6X6X7X7X7X8X8X8X9X9X9EX10X10X10X11X11X11X12X12X12X13X13X13FX14X14X14X15X15X15X16X16X16X17X17X17GX18X18X18X19X19X19X20X20X20HSaved as: BMSC-DNA-2011-2-12123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6Day0(+)-1Day0(+)-1Day0(+)-1CDay0(-)-1Day0(-)-1Day0(-)-1Day3(+)-1Day3(+)-1Day3(+)-1Day3(-)-1Day3(-)-1Day3(-)-1Day6(+)-1Day6(+)-1Day6(+)-1DDay6(-)-1Day6(-)-1Day6(-)-1Day9(+)-1Day9(+)-1Day9(+)-1Day9(-)-1Day9(-)-1Day9(-)-1Day12(+)-1Day12(+)-1Day12(+)-1EDay12(-)-1Day12(-)-1Day12(-)-1Day15(+)-1Day15(+)-1Day15(+)-1Day15(-)-1Day15(-)-1Day15(-)-1Day18(+)-1Day18(+)-1Day18(+)-1FDay18(-)-1Day18(-)-1Day18(-)-1Day21(+)-1Day21(+)-1Day21(+)-1Day21(-)-1Day21(-)-1Day21(-)-1Day24(+)-1Day24(+)-1Day24(+)-1GDay24(-)-1Day24(-)-1Day24(-)-1Day18(+)-2Day18(+)-2Day18(+)-2Day18(-)-2Day18(-)-2Day18(-)-2H2011-4-13DNA Content Assay:1、BMSC-long-termStandards: 6; Blank:1; Samples:17123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6X1X1X1CX2X2X2X3X3X3X4X4X4X5X5X5DX6X6X6X7X7X7X8X8X8X9X9X9EX10X10X10X11X11X11X12X12X12X13X13X13FX14X14X14X15X15X15X16X16X16X17X17X17GHSaved as: BMSC-DNA-2011-4-13123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6Day0(+)-1Day0(+)-1Day0(+)-1CDay3(+)-1Day3(+)-1Day3(+)-1Day3(-)-1Day3(-)-1Day3(-)-1Day6(+)-1Day6(+)-1Day6(+)-1Day6(-)-1Day6(-)-1Day6(-)-1DDay9(+)-1Day9(+)-1Day9(+)-1Day9(-)-1Day9(-)-1Day9(-)-1Day12(+)-1Day12(+)-1Day12(+)-1Day12(-)-1Day12(-)-1Day12(-)-1EDay15(+)-1Day15(+)-1Day15(+)-1Day15(-)-1Day15(-)-1Day15(-)-1Day18(+)-1Day18(+)-1Day18(+)-1Day18(-)-1Day18(-)-1Day18(-)-1FDay21(+)-1Day21(+)-1Day21(+)-1Day21(-)-1Day21(-)-1Day21(-)-1Day24(+)-1Day24(+)-1Day24(+)-1Day24(-)-1Day24(-)-1Day24(-)-1GH2、PDLC-long-termStandards: 6; Blank:1; Samples:19123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6X1X1X1CX2X2X2X3X3X3X4X4X4X5X5X5DX6X6X6X7X7X7X8X8X8X9X9X9EX10X10X10X11X11X11X12X12X12X13X13X13FX14X14X14X15X15X15X16X16X16X17X17X17GX18X18X18X19X19X19HSaved as: PDLC-DNA-2011-4-13123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6Day0(+)Day0(+)Day0(+)CDay3(+)Day3(+)Day3(+)Day3(-)Day3(-)Day3(-)Day6(+)Day6(+)Day6(+)Day6(-)Day6(-)Day6(-)DDay9(+)Day9(+)Day9(+)Day9(-)Day9(-)Day9(-)Day12(+)Day12(+)Day12(+)Day12(-)Day12(-)Day12(-)EDay15(+)Day15(+)Day15(+)Day15(-)Day15(-)Day15(-)Day18(+)Day18(+)Day18(+)Day18(-)Day18(-)Day18(-)FDay21(+)Day21(+)Day21(+)Day21(-)Day21(-)Day21(-)Day24(+)Day24(+)Day24(+)Day24(-)Day24(-)Day24(-)GDay27(+)Day27(+)Day27(+)Day27(-)Day27(-)Day27(-)H2、PDLC-long-termStandards: 6; Blank:1; Samples:21123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6X1X1X1CX2X2X2X3X3X3X4X4X4X5X5X5DX6X6X6X7X7X7X8X8X8X9X9X9EX10X10X10X11X11X11X12X12X12X13X13X13FX14X14X14X15X15X15X16X16X16X17X17X17GX18X18X18X19X19X19X20X20X20X21X21X21HSaved as: PDLC-DNA-2011-4-22123456789101112ABBBS1S1S1S2S2S2S3S3S3BS4S4S4S5S5S5S6S6S6Day0(+)Day0(+)Day0(+)CDay3(+)Day3(+)Day3(+)Day3(-)Day3(-)Day3(-)Day6(+)Day6(+)Day6(+)Day6(-)Day6(-)Day6(-)DDay9(+)Day9(+)Day9(+)Day9(-)Day9(-)Day9(-)Day12(+)Day12(+)Day12(+)Day12(-)Day12(-)Day12(-)EDay15(+)Day15(+)Day15(+)Day15(-)Day15(-)Day15(-)Day18