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From 2011.igem.org

Cyrille

Adding the RFP biobrick in pHM3

We want to insert the biobrick J04450 into pHM3 for three reasons:

• To have a negative control of digestion
• To restore the EXSP biobrick format in the plasmid
• To test the possibility of cloning in this plasmid

3 times 500 ng of the pHM3 plasmid is digested in E P for 30 min

3 times 500ng of J04450 in pSB1C3 is digested using E P sites.

The result is loaded on a gel. The and will undergo a gel purification.

After the gel extraction the yields where very low, so that it was in the noise of the tecan machine. So I decided to pool together the threee tubes of the same serie. Once mixed, vortexed and centrifucated, the concentration was mesures again. The tecan says the concentration is around 10 and 20 ng/mL depending on the tube and on the measure. This is sufficient to carry on.

The ligation was done using the T4 DNA ligase. 5 tubes where prepared as explain:

Tube	1	2	3	4
10x T4 buffer	2µL	2µL	2µL	2µL
T4 DNA ligase	1µL	1µL	1µL	1µL
DNA pHM3 EcoRI-	2µL	2µL	5µL	5µL
J04450	2µL	10µL	5µL	10µL
H2O	13µL	5µL	7µL	2µL