Team:Czech Republic/Interlab study
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Contents
Background
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Design
Materials and methods
Used plasmids
1. Device 1 (20K+21J): J23101 + I13504, in the pSB1C3 backbone
2. Device 2 (22A+21J): J23106 + I13504, in the pSB1C3 backbone
3. Device 3 (22K+21J): J23117 + I13504, in the pSB1C3 backbone
4. Positive control (C+): I20270, in the pSB1C3 backbone
5. Negative control (C-): K823008, in the pSB1C3 backbone
Used strain
E. coli E5alpha
Used material
1. LB-M agar plates with chloramphenicol
2. LB-M agar plates with ampicillin
3. 1,5 ml eppendorf tubes
4. 0,5 ml PCR tubes
5. 50 ml centrifuge conical base and rim tubes
6. NucleoSpin Plasmid DNA, RNA, and protein purification Kit
7. NucleoSpin Gel and PCR Clean-up Kit
8. LB-M medium with chloramphenicol
9. NaOH agarose gel and buffer
10. Sphero Rainbow Calibration Particles, 8 Peaks, 3.0-3.4
Used methods (links)
1. Transformation
2. Miniprep
3. Restriction digest
4. Ligation
5. NucleoSpin Gel Clean-up
6. NucleoSpin Plasmid DNA purification
7. Flow cytometry
Used machines
1. Incubator: Binder, ATP.line CB(E6), CO2 – Incubator with O2 control, model CB160, 230V
2. Orbita Shaker PSU-10i, 280 rpm, 360deg
3. Flow Cytometr C6
4. Thermo-Shaker with cooling for microtubes and PCR plates TS-100C
5. Centrifuge 5424/5424 R
Used software
1. CFlow Plus
2. Microsoft Excel
3. Sphero PMT QC Template
Device | Colony | MEFL | Mean of technical replicates | SD Colony | Mean of device | SD Device | |
---|---|---|---|---|---|---|---|
20K +21J (Device 1) | Colony 1 | I. | 639549 | 657818 | 15504 | 664409 | 29185 |
II. | 677453 | ||||||
III. | 656453 | ||||||
Colony 2 | I. | 689717 | 702990 | 10067 | |||
II. | 714084 | ||||||
III. | 705169 | ||||||
Colony 3 | I. | 620331 | 632419 | 15053 | |||
II. | 623286 | ||||||
III. | 653639 | ||||||
22A+21J (Device 2) | Colony 1 | I. | 481600 | 464304 | 12795 | 392603 | 50712 |
II. | 460265 | ||||||
III. | 451048 | ||||||
Colony 2 | I. | 346283 | 358070 | 9696 | |||
II. | 357896 | ||||||
III. | 370032 | ||||||
Colony 3 | I. | 354544 | 355433 | 4688 | |||
II. | 350188 | ||||||
III. | 361568 | ||||||
22K+21J (Device 3) | Colony 1 | I. | 2635 | 2872 | 220 | 2229 | 454 |
II. | 3165 | ||||||
III. | 2814 | ||||||
Colony 2 | I. | 1786 | 1891 | 74 | |||
II. | 1933 | ||||||
III. | 1953 | ||||||
Colony 3 | I. | 1665 | 1926 | 204 | |||
II. | 1948 | ||||||
III. | 2163 | ||||||
Positive control | Colony 1 | I. | 202361 | 196401 | 5853 | 196401 | / |
II. | 188446 | ||||||
III. | 198395 | ||||||
Negative control | Colony 1 | I. | 224 | 213 | 8 | 213 | / |
II. | 204 | ||||||
III. | 211 |
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