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
Ramon and I prepared the DNA electrophoresis loading buffer, 10 ml of this mixture are enough for thousands of samples.
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
| 15% 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!!!
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|
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