Difference between revisions of "Part:BBa M36689:Experience"

(→‎Applications of BBa_M36689)
(→‎Applications of BBa_M36689)
Line 7: Line 7:
  
  
 +
 +
 +
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.
 +
 +
[[Gel.jpg]]
  
 
Fluorescence Microscopy Images:  
 
Fluorescence Microscopy Images:  
Line 34: Line 48:
 
10. [[File:Before1hr42CExpt.tif]]
 
10. [[File:Before1hr42CExpt.tif]]
 
11. [[File:Mock123am1f3.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:Microscopy_analysis.png]]
 +
 +
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===
 
===Stanford Location===

Revision as of 02:08, 13 December 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

Gel.jpg

Fluorescence Microscopy Images: 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.

Microscopy analysis.png

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

UNIQ7c1c49f5b08e93a8-partinfo-00000000-QINU UNIQ7c1c49f5b08e93a8-partinfo-00000001-QINU