User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/04/21

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Wednesday 21st April 2010

I cultivated the 5 colonies resulting from the Monday transformation in 4ml of liquid lb with 4ul of kanamycin and also a medium control at 9 a.m. I came back a 3 p.m. to extract the plasmid BBa_I712019 (luciferase from Photinus pyralis) with the kit from Roche. For this I centrifuged 3 times the LB to get the whole pellet of bacteria from each tube, alter that I confused the plasmid extraction kit with the PCR exctraction kit, and I resuspended the pellet in 80ul of water, but then I realizad my mistake and centrifugues again to get the pellet, afterwards I extracted the plasmid from each sample using the Roche kit.

Double restriction with EcoR1 and PST:

I prepared a stock for the 5 reactions using the calculations for 6 as it follows:

Reactive....1x [ul]......6x [ul]
DNA..........5..............30
BSA...........1..............6
Buffer2......2.............12
H2O..........10............60
EcoR1........1..............6
PST1..........1..............6
Total..........20...........120

I used the Buffer 2, because it is the one that yields the most efficiency for the 2 enzymes, also remember that the buffer 3 from the kit must be in ice. Finally I put the eppendorfs with the double restriction plasmids in the incubator overnight and the remaining DNA from the plasmid purification in the fridge. Everything is very well labeled.