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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

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!!!