Luận văn : BƯỚC ĐẦU XÂY DỰNG QUY TRÌNH ĐỊNH LƯỢNG CÁC SẢN PHẨM BIẾN ĐỔI GEN BẰNG PHƯƠNG PHÁP REAL-TIME PCR part 10 ppsx

104
104

Gen tham chiếu:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Data

Current Date: 19-Aug-05 12:05 PM
Data generated on: 22-Jul-05 at 06:15 PM.

Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-sua2.odm
Plate Setup file used: GMO-35S.psm
Protocol file used: GMO-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 10:00

Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


Standard Curve Graph for FAM-490



105
105

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 67.5.
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 Ct Ct Set
SQ Mean SD Mean SD
Point

B02 Standard Stan Corn-Ref 1 28.29 4.612 4.10E+04 4.10E+04 N/A 28.29 N/A
B04 Standard Stan Corn-Ref 2 30.26 3.709 5.12E+03 5.12E+03 N/A 30.26 N/A
B06 Standard Stan Corn-Ref 3 32.97 2.806 6.40E+02 6.40E+02 N/A 32.97 N/A
B08 Standard Stan Corn-Ref 4 35.43 1.903 8.00E+01 8.00E+01 N/A 35.43 N/A
B11 Unknown Bt 9 28.93 4.308 2.03E+04 2.03E+04 N/A 28.93 N/A



Đợt 3: Định lƣợng bắp chuyển gen Bt 176 1% (lần 2)
Promoter 35S:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 19-Aug-05 12:17 PM
Data generated on: 16-Aug-05 at 04:58 PM.

Optical data file name: Data 16-Aug-05 1658 taqman dot 3-35S -Bt176.odm
Plate Setup file used: 35S TAQMAN DOT 3.psm
Protocol file used: GMO-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 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


106
106



Standard Curve Graph for FAM-490

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 21.8.
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.

Standard Curve Spreadsheet Data for FAM-490 Units: copy number

Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set
SQ Mean SD Mean SD
Point

E02 Standard ST 35S 5 23.85 3.709 5.12E+03 5.12E+03 N/A 23.85 N/A
E03 Standard ST 35S 6 26.55 2.806 6.40E+02 6.40E+02 N/A 26.55 N/A
E04 Standard ST 35S 7 29.13 1.903 8.00E+01 8.00E+01 N/A 29.13 N/A
E05 Standard ST 35S 8 31.67 1.000 1.00E+01 1.00E+01 N/A 31.67 N/A
F02 Unknown Bt 19 28.56 2.092 1.24E+02 1.24E+02 N/A 28.56 N/A



Gen tham chiếu:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Data

Current Date: 19-Aug-05 12:22 PM
Data generated on: 16-Aug-05 at 04:58 PM.

Optical data file name: Data 16-Aug-05 1658 taqman dot 3-DC Bt 176.odm
Plate Setup file used: 35S TAQMAN DOT 3.psm
Protocol file used: GMO-35S.tmo

Sample volume: 25.00 ul


107

107

Hot Start? No
Well factor collection: Experimental Plate

Comments



Protocol


Cycle 1: ( 1X)
Step 1: 95.0ºC for 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


Standard Curve Graph for FAM-490

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 21.2.
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 Ct Ct Set
SQ Mean SD Mean SD
Point

B02 Standard ST CR 1 23.31 4.612 4.10E+04 4.10E+04 N/A 23.31 N/A
B03 Standard ST CR 2 25.71 3.709 5.12E+03 5.12E+03 N/A 25.71 N/A
B04 Standard ST CR 3 27.04 2.806 6.40E+02 6.40E+02 N/A 27.04 N/A


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108

B05 Standard ST CR 4 30.64 1.903 8.00E+01 8.00E+01 N/A 30.64 N/A
C02 Unknown Bt 3 24.51 4.097 1.25E+04 1.25E+04 N/A 24.51 N/A


Đợt 4: Độ nhạy của phản ứng

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 30-Aug-05 06:46 AM
Data generated on: 22-Jul-05 at 06:15 PM.

Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-do nhay.odm
Plate Setup file used: GMO-35S.psm
Protocol file used: GMO-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 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


Standard Curve Graph for FAM-490


109
109

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 13.7.
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 FAM-490 Units: copy number

Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set
SQ Mean SD Mean SD
Point

E02 Standard Stan Corn-Ref 5 25.73 3.709 5.12E+03 5.12E+03 N/A 25.73 N/A
E04 Standard Stan Corn-Ref 6 28.57 2.806 6.40E+02 6.40E+02 N/A 28.57 N/A
E06 Standard Stan Corn-Ref 7 31.65 1.903 8.00E+01 8.00E+01 N/A 31.65 N/A
E08 Standard Stan Corn-Ref 8 33.12 1.000 1.00E+01 1.00E+01 N/A 33.12 N/A
F08 Unknown Mau 1 copy 4 36.22 0.047 1.11E+00 1.25E+00 1.91E-01 36.09 1.87E-01
F10 Unknown Mau 1 copy 4 35.96 0.141 1.38E+00 1.25E+00 1.91E-01 36.09 1.87E-01



Đợt 5: Định lƣợng các mẫu thí nghiệm (lần 1)

Promoter 35S:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 11-Aug-05 03:43 PM
Data generated on: 22-Jul-05 at 06:15 PM.

Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-sua.odm
Plate Setup file used: GMO-35S.psm
Protocol file used: GMO-35S.tmo

Sample volume: 25.00 ul
Hot Start? No
Well factor collection: Experimental Plate

Comments



Protocol



110
110


Cycle 1: ( 1X)
Step 1: 95.0ºC for 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.

Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


Standard Curve Graph for FAM-490

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 52.2.
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 FAM-490 Units: copy number

Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set
SQ Mean SD Mean SD
Point

E02 Standard Stan Corn-Ref 5 27.43 3.709 5.12E+03 5.12E+03 N/A 27.43 N/A
E04 Standard Stan Corn-Ref 6 30.76 2.806 6.40E+02 6.40E+02 N/A 30.76 N/A
E06 Standard Stan Corn-Ref 7 33.65 1.903 8.00E+01 8.00E+01 N/A 33.65 N/A
E08 Standard Stan Corn-Ref 8 35.89 1.000 1.00E+01 1.00E+01 N/A 35.89 N/A
F02 Unknown B1 5 27.38 3.808 6.42E+03 5.94E+03 6.84E+02 27.49 1.57E-01
F04 Unknown B1 5 27.61 3.737 5.46E+03 5.94E+03 6.84E+02 27.49 1.57E-01
F06 Unknown B2 6 N/A N/A N/A N/A N/A N/A N/A
F08 Unknown B2 6 29.96 2.985 9.65E+02 9.65E+02 N/A 29.96 N/A
G02 Unknown B7 7 27.71 3.704 5.06E+03 4.90E+03 2.23E+02 27.75 6.18E-02

G04 Unknown B7 7 27.80 3.676 4.75E+03 4.90E+03 2.23E+02 27.75 6.18E-02




111
111



Gen tham chiếu:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 11-Aug-05 04:02 PM
Data generated on: 22-Jul-05 at 06:15 PM.

Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-2.odm
Plate Setup file used: GMO-35S.psm
Protocol file used: GMO-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 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


Standard Curve Graph for FAM-490


112
112


Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 72.9.
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 Ct Ct Set
SQ Mean SD Mean SD
Point

B02 Standard Stan Corn-Ref 1 28.55 4.612 4.10E+04 4.10E+04 N/A 28.55 N/A
B04 Standard Stan Corn-Ref 2 30.62 3.709 5.12E+03 5.12E+03 N/A 30.62 N/A
B06 Standard Stan Corn-Ref 3 33.22 2.806 6.40E+02 6.40E+02 N/A 33.22 N/A
B08 Standard Stan Corn-Ref 4 35.84 1.903 8.00E+01 8.00E+01 N/A 35.84 N/A
C02 Unknown B1 1 27.69 4.870 7.42E+04 9.61E+04 3.09E+04 27.42 3.86E-01
C04 Unknown B1 1 27.14 5.072 1.18E+05 9.61E+04 3.09E+04 27.42 3.86E-01
C06 Unknown B2 2 37.03 1.420 2.63E+01 8.38E+01 8.13E+01 36.04 1.40E+00
C08 Unknown B2 2 35.06 2.150 1.41E+02 8.38E+01 8.13E+01 36.04 1.40E+00
D02 Unknown B7 3 28.32 4.636 4.32E+04 4.35E+04 3.33E+02 28.32 9.02E-03
D04 Unknown B7 3 28.31 4.640 4.37E+04 4.35E+04 3.33E+02 28.32 9.02E-03


Đợt 6: Định lƣợng các mẫu thí nghiệm (lần 2)
Promoter 35S:


PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 11-Aug-05 05:04 PM
Data generated on: 03-Aug-05 at 09:21 PM.

Optical data file name: Data 03-Aug-05 2121 TAQMAN DOT 2-35S-MAU.odm

Plate Setup file used: Taqman 35S dot 2 3-8-05.psm
Protocol file used: GMO-35S.tmo

Sample volume: 25.00 ul
Hot Start? No
Well factor collection: Experimental Plate

Comments



Protocol



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113


Cycle 1: ( 1X)
Step 1: 95.0ºC for 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490

Standard Curve Graph for FAM-490

Data Analysis Parameters

Calculated threshold has been replaced by the user selected threshold 65.7.
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 Ct Ct Set
SQ Mean SD Mean SD
Point

E02 Standard St 35S 5-8 5 26.77 3.709 5.12E+03 5.12E+03 N/A 26.77 N/A
E04 Standard St 35S 5-8 6 30.13 2.806 6.40E+02 6.40E+02 N/A 30.13 N/A
E06 Standard St 35S 5-8 7 32.62 1.903 8.00E+01 8.00E+01 N/A 32.62 N/A
E08 Standard St 35S 5-8 8 35.24 1.000 1.00E+01 1.00E+01 N/A 35.24 N/A
F02 Unknown B4 8 26.35 3.920 8.32E+03 7.88E+03 6.32E+02 26.43 1.08E-01
F03 Unknown B4 8 26.51 3.871 7.43E+03 7.88E+03 6.32E+02 26.43 1.08E-01
F04 Unknown B1 9 26.60 3.842 6.95E+03 8.16E+03 1.71E+03 26.39 2.84E-01
F05 Unknown B1 9 26.19 3.972 9.38E+03 8.16E+03 1.71E+03 26.39 2.84E-01
F06 Unknown B2 10 28.93 3.085 1.22E+03 9.45E+02 3.85E+02 29.33 5.63E-01
F07 Unknown B2 10 29.73 2.828 6.73E+02 9.45E+02 3.85E+02 29.33 5.63E-01
F08 Unknown B6 11 34.16 1.395 2.48E+01 3.33E+01 1.20E+01 33.81 4.95E-01
F09 Unknown B6 11 33.46 1.622 4.18E+01 3.33E+01 1.20E+01 33.81 4.95E-01

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114

F10 Unknown B7 12 27.36 3.594 3.93E+03 4.16E+03 3.22E+02 27.29 1.04E-01
F11 Unknown B7 12 27.21 3.642 4.39E+03 4.16E+03 3.22E+02 27.29 1.04E-01
G02 Unknown B10 13 26.05 4.019 1.05E+04 7.24E+03 4.56E+03 26.69 9.09E-01
G03 Unknown B10 13 27.33 3.603 4.01E+03 7.24E+03 4.56E+03 26.69 9.09E-01
G04 Unknown B11 14 24.99 4.362 2.30E+04 1.77E+04 7.47E+03 25.40 5.83E-01
G05 Unknown B11 14 25.81 4.095 1.25E+04 1.77E+04 7.47E+03 25.40 5.83E-01






Gen tham chiếu:

PCR Standard Curve Report
with PCR Amp Cycle Graph
PCR Base Line Subtracted Curve Fit Data

Current Date: 20-Aug-05 03:17 PM
Data generated on: 03-Aug-05 at 09:21 PM.

Optical data file name: Data 03-Aug-05 2121 TAQMAN DOT 2-GEN DC.odm
Plate Setup file used: Taqman 35S dot 2 3-8-05.psm
Protocol file used: GMO-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 10:00
Cycle 2: ( 45X)
Step 1: 95.0ºC for 00:15
Step 2: 60.0ºC for 01:00
Data collection and real-time analysis enabled.
Cycle 3: ( 1X)
Step 1: 4.0ºC HOLD

PCR Amp/Cycle Graph for FAM-490


115
115



Standard Curve Graph for FAM-490

Data Analysis Parameters

Calculated threshold using the maximum correlation coefficient approach is 14.3.
User selected baseline cycles are 2 to 19.
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 Ct Ct Set
SQ Mean SD Mean SD
Point

B02 Standard St CR 1-4 1 22.19 4.612 4.10E+04 4.10E+04 N/A 22.19 N/A
B04 Standard St CR 1-4 2 24.81 3.709 5.12E+03 5.12E+03 N/A 24.81 N/A
B06 Standard St CR 1-4 3 26.55 2.806 6.40E+02 6.40E+02 N/A 26.55 N/A
B08 Standard St CR 1-4 4 30.57 1.903 8.00E+01 8.00E+01 N/A 30.57 N/A
C02 Unknown B4 1 23.02 4.268 1.85E+04 1.29E+04 7.98E+03 23.63 8.57E-01
C03 Unknown B4 1 24.23 3.861 7.26E+03 1.29E+04 7.98E+03 23.63 8.57E-01
C04 Unknown B1 2 25.69 3.371 2.35E+03 2.59E+03 3.40E+02 25.57 1.70E-01
C05 Unknown B1 2 25.45 3.452 2.83E+03 2.59E+03 3.40E+02 25.57 1.70E-01
C06 Unknown B2 3 25.60 3.403 2.53E+03 4.46E+03 2.73E+03 25.00 8.47E-01
C07 Unknown B2 3 24.40 3.806 6.40E+03 4.46E+03 2.73E+03 25.00 8.47E-01
C08 Unknown B6 4 23.91 3.970 9.33E+03 9.27E+03 7.56E+01 23.92 1.05E-02
C09 Unknown B6 4 23.93 3.965 9.22E+03 9.27E+03 7.56E+01 23.92 1.05E-02
C10 Unknown B7 5 22.55 4.426 2.67E+04 1.52E+04 1.62E+04 23.82 1.79E+00
C11 Unknown B7 5 25.09 3.575 3.76E+03 1.52E+04 1.62E+04 23.82 1.79E+00
D02 Unknown B10 6 24.92 3.630 4.26E+03 4.27E+03 1.07E+01 24.92 3.23E-03
D03 Unknown B10 6 24.92 3.631 4.28E+03 4.27E+03 1.07E+01 24.92 3.23E-03
D04 Unknown B11 7 30.59 1.726 5.32E+01 3.31E+02 3.93E+02 29.01 2.23E+00
D05 Unknown B11 7 27.44 2.785 6.09E+02 3.31E+02 3.93E+02 29.01 2.23E+00