Team:Bielefeld-CeBiTec/Notebook/Miscellaneous
Miscellaneous
- fluorescence imaging
- we took lysate with sfGFP, lysis buffer and purified GFP
- We got a filter from light engineering from the women cultural center Bielefeld e.V. ("Frauenkulturzentrum Bielefeld e.V.") and put it in front of the smartphone.
- the picture showed great evidence for possible fluorescence imaging with the right filters
This Week we started with the iGEM 2015 Measurement Interlab Study:
- Heat shock transformation of BioBricks from the distribution:
- BBa_K823005 (Anderson promoter J23101)
- BBa_K823008 (Anderson promoter J23106)
- BBa_K823013 (Anderson promoter J23117)
- BBa_I13504 (GFP)
- we made a preselection on basis of the light transmitting spectra of the filters, we bought
- we testet DEEP PURPLE BLUE (797), MEDIUM PURPLE (049) MAUVE (126), CHRYSALIS PINK (798), CONGO BLUE (181), ULTIMATE VIOLET (707), SPECIAL KH LAVENDER (799), TOKYO BLUE (071), J WINTER BLUE (713), PALACE BLUE (198), DEEP BLUE (120) and SPECIAL MEDIUM BLUE (363) as possible filters in front of the flash
- As possible filter in front of the camera we tested VELVET GREEN (735), FLUORESCENT GREEN (219), PRIMARY GREEN (139), DARK YELLOW GREEN (090) and TWICKENHAM GREEN (736)
- we filled reactions tubes with lysis buffer, lysate without sfGFP, sfGFP lysate and purified GFP, each tube were filled with 500 µL and we took an empty reaction tube.
- to get pictures under same conditions we took a carton and put black carton in it, to get an dark environment
- we took pictures of the tube with the different filter combinations
- afterwards we analyzed the pictues with the image processing software Fiji
- we draw a square in the pictures and took it to the relevant places where we wanted to measure the fluorescence
- you can get the measurement results from fiji and calculate the fluorescence with the following formula: fluorescence = Integrated Density – (Area of selected X Mean fluorescence of background readings)
- we took the results and analyzed them and made the conclusion that, twickenham green in combination with tokyo blue is the best combination to photograph gfp with your smartphone with less background as possible
- fluorescence imaging of GFP
iGEM 2015 Measurement Interlab Study:
- Colonies from the BioBrick Transformation were used to inoculate over night cultures
- 5 ml LB-Medium + Cm (20 µg/ml), 37°C
- Mini-preps of KRX(BBa_K823005), KRX(BBa_K823008) and KRX(BBa_K823013)
- Plasmids stored at -20°C
- Second heat shock transformation of chem. competent KRX with BBa_I13504
- Colonies were used to inoculate over night cultures (5 ml LB + Cm20, 37°C)
- we made a preselection on basis of the light transmitting spectra of the filters, we bought
- DEEP ORANGE (158), DEEP GOLDEN AMBER (135), TERRY RED (781), MADGE (507), FIRE (019), FLAME RED (164), LIGHT RED (182) and PRIMARY RED (106) as possible filters in front of the flash
- As possible filter in front of the camera we tested PRIMARY GREEN (139), DARK YELLOW GREEN (090), AURORA BOREALIS GREEN (740) and TWICKENHAM GREEN (736)
- we filled reactions tubes with lysis buffer, lysate without mRFP and mRFP lysate each tube were filled with 500 µL and we took an empty reaction tube.
- to get pictures under same conditions we took a carton as in the week before
- we took pictures of the tube with the different filter combinations
- afterwards we analyzed the pictues with the image processing software Fiji
- we draw a square in the pictures and took it to the relevant places where we wanted to measure the fluorescence
- you can get the measurement results from fiji and calculate the fluorescence with the following formula: fluorescence = Integrated Density – (Area of selected X Mean fluorescence of background readings)
- we took the results and analyzed them and made the conclusion that, twickenham green in combination with light red is the best combination to photograph mRFP with your smartphone with less background as possible
- fluorescence imaging of mRFP
iGEM 2015 Measurement Interlab Study:
- Mini-prep of KRX(BBa_I13504)
- Plasmid stored at -20°C