Team:TU Delft/23 June 2010 content

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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 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!!!