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Team:University of Ottawa/31 July 2008
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Today in the Lab
Chris
Microwave Digestion Experiment
- Following protocol outlined online, tested a microwave protocol to hasten digestion period.
- Digested DQ232597 with HindIII. Ran five samples: 5 seconds in micro, 10 seconds, 15 seconds, 10 seconds in and 2 minutes out (four times), water control, water control 10 seconds in microwave and 2 minutes out, 1 hr traditional digest.
- Denatured HindIII at 80 C for 20 minutes, then ran products on a gel.
- Results promising; however, more experimentation needed to confirm validity of this protocol
Matt
Ligation
- Another ligation of PTP2 insert with pSSA42 vector was performed with 3:1 ratio, 6:1 ratio, Vector with ligase, vector without ligase and H2O control.
- 2 hr ligation was unsucessful, overnight ligation was usccessful for both 3:1 and 6:1.
Transformation
- Transformed ligation product into competent cells - 3 different plates -3:1, 6:1 and control.
Dan
Gel Confirmation
- One gel was run for the confirmation of the ligation of constructs 0A 0B and T, this gel showed that the ligation was successful
Gel Extraction
- The successful gel indicated that I could go forward and run all the samples on a gel for gel extraction (ran over 6 wells) and the correct bands were extracted.
Digestion
- 2 samples were created (one for gel confirmation and one for transformation), both were digested with ClaI
PCR cleanup
- PCR cleanup of the digestion was performed in order to prevent salts from inhibiting gel electrophoresis.
Ligation
- Ligation was run overnight in the thermocycler of pSSA140B and p1T(0A0B)
