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91 91 Phụ lục 6: Dung dịch ethanol 70%. - C 2 H 5 OH 70 %. Phụ lục 7: Potassium acetate 3 M, pH 5,2 - Potassium acetate 3 mol/l - Hòa tan trong nước siêu sạch. - Hấp tiệt trùng - Bảo quản ở nhiệt độ phòng. Phụ lục 8: Kết quả đo OD các quy trình ly trích Kết quả thí nghiệm lần 1: Quy trình Thông số Mẫu 1 Mẫu 2 Mẫu 3 Mẫu 4 1 OD 260nm /OD 280nm 1.524 1.88 1.305 1.679 Nồng độ DNA (µg) 1.824 1.416 1.683 1.923 2 OD 260nm /OD 280nm 1.888 1.678 1.825 1.731 Nồng độ DNA (µg) 6.06 3.135 5.787 3.276 3 OD 260nm /OD 280nm 1.84 2.08 1.68 1.8 Nồng độ DNA (µg) 10.626 8.769 15.237 10.284 Kết quả thí nghiệm lần 2: Quy trình Thông số Mẫu 1 Mẫu 2 Mẫu 3 Mẫu 4 1 OD 260nm /OD 280nm 1.254 1.73 1.872 1.847 Nồng độ DNA (µg) 2.208 1.017 1.539 1.191 2 OD 260nm /OD 280nm 1.848 1.875 1.696 1.594 Nồng độ DNA (µg) 7.674 6.756 5.856 4.389 3 OD 260nm /OD 280nm 1.86 1.78 2.1 1.96 Nồng độ DNA (µg) 13.155 11.559 11.205 10.803 92 92 Kết quả thí nghiệm lần 3: Quy trình Thông số Mẫu 1 Mẫu 2 Mẫu 3 Mẫu 4 1 OD 260nm /OD 280nm 1.815 1.509 1.667 1.864 Nồng độ DNA (µg) 1.503 2.286 2.691 1.73 2 OD 260nm /OD 280nm 1.245 1.208 1.755 1.697 Nồng độ DNA (µg) 4.134 5.022 3.375 5.0505 3 OD 260nm /OD 280nm 1.81 1.84 1.79 1.82 Nồng độ DNA (µg) 10.767 10.911 11.268 11.352 93 93 Phụ lục 9: Kết quả phản ứng Real-Time PCR với thuốc nhuộm SYBR Green I Đợt 1: PCR Quantification with Melt Curve Report PCR Base Line Subtracted Curve Fit Data Current Date: 25-Aug-05 06:24 PM Data generated on: 28-Jul-05 at 12:47 PM. Optical data file name: Data 28-Jul-05 1247-SYBR Green 35S-GMO.odm Plate Setup file used: SYBR-Green 35S.psm Protocol file used: SYBR Green-35S.tmo Sample volume: 25.00 ul Hot Start? No Well factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) Step 1: 95.0ºC for 03:00 Cycle 2: ( 50X) Step 1: 95.0ºC for 00:25 Step 2: 62.0ºC for 00:30 Step 3: 72.0ºC for 00:45 Data collection and real-time analysis enabled. Cycle 3: ( 1X) Step 1: 72.0ºC for 07:00 Cycle 4: ( 80X) Step 1: 55.0ºC for 00:10 Increase setpoint temperature after cycle 2 by 0.5ºC Melt curve data collection and analysis enabled. Cycle 5: ( 1X) Step 1: 4.0ºC HOLD PCR Amp/Cycle Graph for SYBR-490 94 94 Data Analysis Parameters Calculated threshold using the maximum curvature approach is 20.0. Per-well baseline cycles have been determined automatically. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. PCR Quantification Spreadsheet Data for SYBR-490 Well Identifier Ct Setpoint D02 Bt-176 1% 27.56 D10 N/A Melt Curve Graph for SYBR-490 Melt Curve Analysis Parameters Weighted Mean digital filtering has been applied. Global filtering is off. Threshold for automatic peak detection is set at 1.00. Melt Curve Analysis Spreadsheet Data for SYBR-490 Well Well Identifier Peak ID Melt Temp Beg. Temp End Temp Peak Descriptor D2 Bt-176 1% D2.1 86.5 80.5 91.5 D2.2 77.0 74.5 80.0 D2.3 73.0 72.5 74.0 D2.4 70.0 69.5 72.5 D2.5 68.0 66.5 69.0 D2.6 64.5 57.0 66.0 D10 D10.1 60.0 55.0 62.0 95 95 Đợt 2: PCR Quantification with Melt Curve Report PCR Base Line Subtracted Curve Fit Data Current Date: 25-Aug-05 06:29 PM Data generated on: 05-Aug-05 at 03:35 PM. Optical data file name: Data 05-Aug-05 1505 SYBR Green -dot 5-3.odm Plate Setup file used: SYBR Green 35S dot 5 5-8-0518.psm Protocol file used: SYBR Green-35S-2.tmo Sample volume: 25.00 ul Hot Start? No Well factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) Step 1: 95.0ºC for 01:30 Cycle 2: ( 50X) Step 1: 95.0ºC for 00:25 Step 2: 62.0ºC for 00:30 Step 3: 72.0ºC for 00:45 Data collection and real-time analysis enabled. Cycle 3: ( 1X) Step 1: 72.0ºC for 07:00 Cycle 4: ( 80X) Step 1: 55.0ºC for 00:10 Increase setpoint temperature after cycle 2 by 0.5ºC Melt curve data collection and analysis enabled. Cycle 5: ( 1X) Step 1: 15.0ºC HOLD PCR Amp/Cycle Graph for SYBR-490 Data Analysis Parameters 96 96 Calculated threshold has been replaced by the user selected threshold 68.8. User selected baseline cycles are 2 to 10. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. PCR Quantification Spreadsheet Data for SYBR-490 Well Identifier Ct Setpoint D02 B1 20.62 F07 B10 24.04 D06 B11 41.04 D04 B2 16.96 B04 B3 N/A D10 B4 22.22 B06 B5 N/A F03 B6 21.15 F05 B7 22.22 F09 B9 N/A Melt Curve Graph for SYBR-490 Melt Curve Analysis Parameters Weighted Mean digital filtering has been applied. Global filtering is off. Threshold for automatic peak detection is set at 1.00. Melt Curve Analysis Spreadsheet Data for SYBR-490 Well Well Identifier Peak ID Melt Temp Beg. Temp End Temp Peak Descriptor B4 B3 B4.1 62.5 60.5 68.0 B4.2 59.0 55.5 60.5 B6 B5 B6.1 91.5 89.5 93.5 B6.2 86.0 85.5 88.0 B6.3 83.0 77.5 85.5 B6.4 75.0 74.0 77.0 B6.5 71.0 69.0 73.0 B6.6 67.5 66.5 68.5 B6.7 64.0 62.5 66.0 B6.8 60.5 56.0 62.0 D2 B1 D2.1 86.5 78.0 91.5 D2.2 69.0 67.5 70.0 D2.3 66.5 64.5 67.0 D2.4 62.5 62.0 63.5 D2.5 59.5 56.5 61.0 D4 B2 D4.1 93.5 90.0 94.5 D4.2 86.5 82.5 89.5 97 97 D4.3 81.5 78.5 82.0 D4.4 74.0 73.0 77.0 D4.5 71.0 69.5 72.0 D4.6 67.0 65.0 69.0 D4.7 62.0 60.0 62.5 D6 B11 D6.1 92.5 92.0 94.5 D6.2 86.0 81.0 91.0 D6.3 79.0 76.0 80.0 D6.4 73.5 73.0 75.5 D6.5 71.0 69.5 72.0 D6.6 68.0 67.0 69.0 D6.7 65.0 61.0 66.5 D6.8 59.5 56.5 60.5 D10 B4 D10.1 86.5 82.0 91.0 D10.2 79.0 77.0 81.5 D10.3 72.0 70.0 73.5 D10.4 67.0 65.5 69.0 D10.5 64.5 61.0 65.0 D10.6 60.0 57.0 61.0 F3 B6 F3.1 86.0 82.0 89.5 F3.2 79.5 77.5 81.5 F3.3 73.5 71.5 75.5 F3.4 69.0 65.5 70.5 F3.5 63.0 62.0 65.0 F3.6 59.5 55.0 60.5 F5 B7 F5.1 86.0 79.0 92.5 F5.2 77.0 75.0 77.5 F5.3 72.0 71.0 74.5 F5.4 65.0 62.5 69.0 F5.5 59.5 55.5 62.0 F7 B10 F7.1 86.0 77.5 90.0 F7.2 72.5 71.0 74.5 F7.3 67.5 66.0 70.5 F7.4 63.5 62.0 65.5 F7.5 60.5 56.5 61.5 F9 B9 F9.1 83.0 81.0 90.0 F9.2 78.0 76.0 80.5 F9.3 74.0 73.5 75.0 F9.4 72.0 70.0 73.0 F9.5 66.5 64.0 68.5 F9.6 62.0 55.0 63.5 98 98 Đợt 3: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 29-Aug-05 04:11 PM Data generated on: 17-Aug-05 at 03:21 PM. Optical data file name: Data 17-Aug-05 1520 SYBR Dot 6-2-bao cao.odm Plate Setup file used: SYBR Green I dot 6 17-7-0522.psm Protocol file used: SYBR Green-35S-2.tmo Sample volume: 25.00 ul Hot Start? No Well factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) Step 1: 95.0ºC for 01:30 Cycle 2: ( 50X) Step 1: 95.0ºC for 00:25 Step 2: 62.0ºC for 00:30 Step 3: 72.0ºC for 00:45 Data collection and real-time analysis enabled. Cycle 3: ( 1X) Step 1: 72.0ºC for 07:00 Cycle 4: ( 80X) Step 1: 55.0ºC for 00:10 Increase setpoint temperature after cycle 2 by 0.5ºC Melt curve data collection and analysis enabled. Cycle 5: ( 1X) Step 1: 15.0ºC HOLD PCR Amp/Cycle Graph for SYBR-490 99 99 Standard Curve Graph for SYBR-490 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 18.2. User selected baseline cycles are 2 to 24. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for SYBR-490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point B02 Standard ST 35S 1 22.99 3.709 5.12E+03 5.12E+03 N/A 22.99 N/A B03 Standard ST 35S 2 24.32 2.806 6.40E+02 6.40E+02 N/A 24.32 N/A B04 Standard ST 35S 3 25.26 1.903 8.00E+01 8.00E+01 N/A 25.26 N/A D02 Unknown Mau 1 copy 2 27.58 0.106 1.28E+00 8.88E+00 1.08E+01 26.88 9.86E-01 F02 Unknown Mau 1 copy 2 26.18 1.217 1.65E+01 8.88E+00 1.08E+01 26.88 9.86E-01 Melt Curve Graph for SYBR-490 100 100 Melt Curve Analysis Parameters Weighted Mean digital filtering has been applied. Global filtering is off. Threshold for automatic peak detection is set at 1.00. Melt Curve Analysis Spreadsheet Data for SYBR-490 Well Well Identifier Peak ID Melt Temp Beg. Temp End Temp Peak Descriptor B2 ST 35S B2.1 86.0 80.0 90.0 B2.2 74.5 74.0 76.5 B2.3 73.0 70.5 73.5 B2.4 68.0 66.5 70.0 B2.5 65.0 64.0 66.0 B2.6 58.5 55.0 63.5 B3 ST 35S B3.1 86.0 76.0 89.5 B3.2 73.5 73.0 75.5 B3.3 69.5 68.5 72.5 B3.4 65.5 64.0 68.0 B3.5 58.5 55.0 61.5 B4 ST 35S B4.1 86.5 79.0 91.0 B4.2 77.0 75.0 78.5 B4.3 71.5 70.0 74.5 B4.4 68.0 66.5 69.5 B4.5 64.5 61.5 65.5 B4.6 59.0 56.5 61.0 D2 mau 1 copy D2.1 86.5 77.5 91.0 D2.2 75.0 73.0 76.0 D2.3 71.5 70.5 72.5 D2.4 67.0 66.0 69.5 D2.5 58.5 55.5 61.0 F2 mau 1 copy F2.1 85.5 76.0 90.0 F2.2 73.5 70.5 75.5 F2.3 68.0 67.0 70.0 F2.4 65.0 64.5 66.5 F2.5 62.5 61.5 64.0 F2.6 58.5 56.5 61.0 [...]... 2: Định lƣợng bắp chuyển gen Bt 176 1% (lần 1) Promoter 35S: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Data Current Date: Data generated on: 19- Aug-05 1 2:0 1 PM 22-Jul-05 at 0 6:1 5 PM Optical data file name: Plate Setup file used: Protocol file used: Data 22-Jul-05 1815-Truong-35S-dot 1-sua2.odm GMO-35S.psm GMO-35S.tmo Sample volume: Hot Start? Well factor collection:... GMO-35S.tmo Sample volume: Hot Start? Well factor collection: 25.00 ul No Experimental Plate Comments Protocol Cycle 1: ( 1X) Step 1: 95 .0ºC Cycle 2: ( 45X) Step 1: 95 .0ºC Step 2: 60.0ºC Data collection and real-time analysis enabled Cycle 3: ( 1X) Step 1: 4.0ºC for 1 0:0 0 for 0 0:1 5 for 0 1:0 0 HOLD PCR Amp/Cycle Graph for FAM- 490 Standard Curve Graph for FAM- 490 101 102 Data Analysis Parameters Calculated threshold... Phụ lục 1 0: Kết quả chạy phản ứng Real-Time PCR với mẫu dò Taqman Đợt 1: Đƣờng chuẩn p35S PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Data Current Date: Data generated on: 20-Aug-05 0 6:1 7 PM 22-Jul-05 at 0 6:1 5 PM Optical data file name: Plate Setup file used: Protocol file used: Data 22-Jul-05 1815-Truong-35S-dot 1-sua2.odm GMO-35S.psm GMO-35S.tmo Sample volume: Hot Start?... factor collection: 25.00 ul No Experimental Plate Comments Protocol Cycle 1: ( 1X) 102 Set 103 Step 1: 95 .0ºC Cycle 2: ( 45X) Step 1: 95 .0ºC Step 2: 60.0ºC Data collection and real-time analysis enabled Cycle 3: ( 1X) Step 1: 4.0ºC for 1 0:0 0 for 0 0:1 5 for 0 1:0 0 HOLD PCR Amp/Cycle Graph for FAM- 490 Standard Curve Graph for FAM- 490 Data Analysis Parameters Calculated threshold has been replaced by the... coefficient approach is 33 .9 User selected baseline cycles are 2 to 20 Data analysis window is set at 95 .00% of a cycle, centered at end of the cycle Weighted Mean digital filtering has been applied Global filtering is off Standard Curve Spreadsheet Data for FAM- 490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Mean SQ SD Ct Mean Ct SD 26.83 30.04 33.07 3.7 09 2.806 1 .90 3 5.12E+03 6.40E+02 8.00E+01... baseline cycles are 2 to 20 Data analysis window is set at 95 .00% of a cycle, centered at end of the cycle Weighted Mean digital filtering has been applied Global filtering is off Standard Curve Spreadsheet Data for FAM- 490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Mean SQ SD Ct Mean Ct SD 27.88 31.35 34.25 36.22 32. 59 3.7 09 2.806 1 .90 3 1.000 2.302 5.12E+03 6.40E+02 8.00E+01 1.00E+01 2.00E+02... SD 27.88 31.35 34.25 36.22 32. 59 3.7 09 2.806 1 .90 3 1.000 2.302 5.12E+03 6.40E+02 8.00E+01 1.00E+01 2.00E+02 5.12E+03 6.40E+02 8.00E+01 1.00E+01 2.00E+02 N/A N/A N/A N/A N/A 27.88 31.35 34.25 36.22 32. 59 N/A N/A N/A N/A N/A Point E02 E04 E06 E08 E11 Standard Standard Standard Standard Unknown Stan Corn-Ref Stan Corn-Ref Stan Corn-Ref Stan Corn-Ref Bt 5 6 7 8 10 103 Set . collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) Step 1: 95 .0ºC for 0 1:3 0 Cycle 2: ( 50X) Step 1: 95 .0ºC for 0 0:2 5 Step 2: 62.0ºC for 0 0:3 0 Step 3: 72.0ºC for 0 0:4 5. Cycle 2: ( 50X) Step 1: 95 .0ºC for 0 0:2 5 Step 2: 62.0ºC for 0 0:3 0 Step 3: 72.0ºC for 0 0:4 5 Data collection and real-time analysis enabled. Cycle 3: ( 1X) Step 1: 72.0ºC for 0 7:0 0 Cycle. Step 1: 95 .0ºC for 0 0:2 5 Step 2: 62.0ºC for 0 0:3 0 Step 3: 72.0ºC for 0 0:4 5 Data collection and real-time analysis enabled. Cycle 3: ( 1X) Step 1: 72.0ºC for 0 7:0 0 Cycle 4: ( 80X)