Team:TU Delft/23 June 2010 content
From 2010.igem.org
Lab work
Characterization of Anderson RBS sequences
Ligations were performed using the overnight digested BioBricks. The following ligation reactions was performed:
# | BioBrick | Fragment 1 | Fragment 2 | Recipient vector |
1 | 4 μL μL ‘E–J1100–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
2 | 4 μL μL ‘E–J1101–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
3 | 4 μL μL ‘E–J1107–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
4 | 4 μL μL ‘E–J1117–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
5 | 4 μL μL ‘E–J1127–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E-linear pSB1T3–P’ | |
6 | negative control | - | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ |
We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added. The mixtures were incubated overnight at 16 °C.
Media and solutions
By Hugo
The recipe is as follows:
Compound | Amount required | Final concentration |
Bromophenol blue | 0.025 g | 0.25% w/v |
Xylene Cyanol FF | 0.025 g | 0.25% w/v |
Ficoll (type 400: Pharmacia) | 1.5 g | 0.25% w/v |
Water | As much as you need for 10 ml |
WARNING: THE POWDERS ARE ELECTROSTATIC. WEAR GLOVES AND CLEAN ALL THE PLACE WITH ALCOHOL (70% v/v)... SERIOUSLY, THE BLUE STUFF IS EVERYWHERE!!!