Part:BBa_M36689:Experience

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Applications of BBa_M36689

RT-PCR Gel: Wells: Using a KB+ ladder I: Plate A, "Mock transfection", negative control II: Plate AA, Experimental HS-Rec Plasmid III: Plate B, Exprimental HS-Rec Plasmid IV: Plate C, Experimental HS-Rec Plasmid V: Plate D, Experimental HS-Rec Plasmid VI: Plate F, Postitive control pComet plasmid All wells showed a strong band at 170 bp, which corresponds to the expected band size. However, presence of the same band in all 6 wells, including well I which had no plasmid and well VI, which had the pocket plasmid, suggests contamination or other unexpected result.

Fluorescence Microscopy Images: Fluorescence Microscopy was performed using 40% exposure on fluorescence and 39% exposure on visible light spectra. Overall, a minimum of 1 picture was taken of each negative control mock transfection well, and positive control well, and 6 pictures were taken of each experimental well, to ensure that fluorescence microscopy could be accurately measured. A representative sample of this microscopy data is shown below, but the full catalogue of microscopy images was too large to upload. Contact rfuisz@stanford.edu for complete data, which is available as a zip file.

1: Post Heat shock, 1 hr 42˚C HS-Rec Plasmid 2: Post Heat shock, 1 hr 45˚C HS-Rec Plasmid 3: Post Heat shock, 2 hr 42˚C HS-Rec Plasmid 4: Post Heat shock, 2 hr 42˚C pComet Postive Control 5: Post Heat shock, 2 hr 45˚C HS-Rec Plasmid 6: Pre Heat shock, 2 hr 45˚C HS-Rec Plasmid 7: Pre Heat shock, 1 hr 45˚C HS-Rec Plasmid 8: Pre Heat shock, 2 hr 42˚C HS-Rec Plasmid 9: Pre Heat shock, 2 hr 45˚C pComet Positive Control 10: Pre Heat shock, 1 hr 42˚C HS-Rec Plasmid 11: Pre Heat shock, 2 hr 42˚C Mock transfection Negative Control

1. File:After1hr42CExpt.tif 2. File:After1hr45CExpt124ce1f5.tif 3. File:After2hr42CExpt.tif 4. File:After+2hr42C 124bp1f1.tif 5. File:After2hrs45CExpt 124de2f5.tif 6. File:Before2hr45CExpt 123de2f4.tif 7. File:Before1hr45CExpt 123ce2f3a.tif 8. File:Before2hr4cCExpt.tif 9. File:Before+2hr42C 12.3.bp1f1.tif 10. File:Before1hr42CExpt.tif 11. File:Mock123am1f3.tif

Fluorescence Microscopy Analysis For each image taken using the GFP filter set, we analyzed individual cells exhibiting fluorescence and analyzed their intensities using Image J Software. In order to quantitatively determine this, we adjusted for corrected total cell fluorescence (CTCF) using the following formula: CTCF = Integrated Density - (Area of Cell * Mean Background Fluorescence Reading). This was done for each heat shock experimental sample condition for our “Experimental” samples. These values were then averaged. We used a two-tailed, unpaired t test to determine significance. File:SARF Microscopy Image J analysis

Plate Reader Data On our second experimental transfection, cells were trypsinized 24 hours after heat shock and transferred to a 96 well plate for fluorescence assays. Analysis of this data suggests that data is inconclusive- this is likely attributable to the low transfection efficiency, and the difficulty of identifying quantitative increases in fluorescence using somewhat noisy data. File:Plate Reader Assay.xls

Stanford Location

Barcode #: 0128166602

Plasmid Name: HS-Rec

Antibiotic Resistance: Ampicillin

DNA 2.0 gene #:193566

Organism Expressed in: HeLa

Sensor/Actuator: Both

User Reviews

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