User:Nelson Augusto Berrocal/Notebook/WiFi Coli 2010 Wet Lab/2010/08/02

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August 02nd, 2010

This week I'm gonna make a ligation with LuxAB, I'll try to finish the ligation during the week and make the first luminescence trials; today I made a restriction of LuxAB with EcoRI and PstI, then I inactivated the enzyme from the samples and let the ligation incubating at 37°C overnight.

I'm gonna make two ligations, one with pSB1C3 and the other with plasmid pSB11k5(+)73 from Stratagene following the next restriction protocol:
Cutting plasmid pSB1C3
Restriction 5μL in 20μL with EcoRI and PstI.

Dilute 5μL in 20μL and then if the gel shows that it's cutted I'll use 1μL of this mix in the ligation.
Ligation:
10μL total
5μL insert
1μL vector
1μL buffer
1μL enzyme
2μL H20


Restriction of plasmid pSB11k5(+)73 from Miguel
Dilute 5μL in 20μL and then if the gel shows that it's cutted I'll use 1μL of this mix in the ligation.
2μL buffer
1μL BSA
2μL each restriction enzyme
10μL H20


LuxAB Restriction 10μL total.
5μL insert (LuxAB)
1μL buffer
1μL BSA
1μL each enzyme
1μL H20

I used NE buffer 3 for restrictictions.