ETHZ/Lab book
From 2007.igem.org
Contents |
Mon, 13. Aug. 2007
- Markus and Martin started labwork at 3pm.
- Cutting out the parts from the minipreped DNA ( ) with the restriction enzymes EcoRI and PstI:
Prepared 4 ED with numbering 11-14
Poored in each: 0.1 µl BSA, 0.5 µl EcoRI, 0.5 µl PstI, 1.0 µl Buffer, 2.9 µl steriled water
Poored in the different EDs: 5 µl of the uncut DNA from #11-14
Incubated it for ~ 90 min (Andy said that if we want to use it the same day, then we should incubate it for at least 1 h, but several hours are better, overnight incubation is best...) - Preparing the Agarose Gel for the electrophoresis:
Decided to make a 0.8 % Agarose gel.
Weighted 640 µg of Agarose in an 100 ml Erlenmeyer Flask and mixed it with 80 ml 1xTAE and putted the mixture for 5 min in the microwave on 320 W - the whole Agarose was dissolved.
Waited until the solution was handwarm an then poored it in and waited several minutes until the agarose gel became "solid". - Preparing the overnight culture of E.coli Top10 cells for the preparation of competent cells on tuesday:
Prepared a sterile Falcon tube.
Filled in 5ml of LB liquid in the tube.
Streaked over a dish of E.coli Top10 with the sterile tip of a pipete (Andy told us to do so, if we haven't got sterile toothpicks) and throwed it in the FT.
Started incubation over night in the shaking incubator. - Christos and Tim joined
- Prepared the electrophoresis dish and the cut DNA, the uncut DNA (Andy told us to let run both on the same gel) and the marker:
Putted the "solid" gel in the electrophoresis dish and filled it up with 1xTAE until the whole gel was covered.
Prepared 10 times (8 for the DNA and 2 for the markers) 3 µl of loading buffer on parafilm.
Added twice the marker lambda (3µl), 4 times 5µl of the uncut DNA and 4 times 10 µl of the cut DNA (Andy told us all the amounts).
Pipetted the DNA in the gel, with one marker on the left side and one marker on the right side.
Started the electrophoresis and left it running for ~30 min.
Put the gel in the EtBr for 5 min and then in the Kodak scanner.
Didn't get a clear result, so we putted the gel back in the EtBr for ~15 min, but the results in the Kodak scanner and still haven't clear results. Andy said, that there was probably a mistake in one of the earlier steps (miniprep) and that we should make new competent cells...
Tue, 14. Aug. 2007
Morning shift from 9 am:
- Prepared competent cells.
The aliquots are stored in the frezer -80°C in the basement, slot 17.
Evening shift from 5 pm:
- Transformation of J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053, they are in the 37°C shaking incubator.
The uncut DNA is stored in the freezer of Andy's fridge, the lowest drawer, in a green rack. - Made new Liquid LB and LB Agar (they are at the autoclave)
- Made some Agarose Gel for the Electrophoresis on Wednesday.
One for the small Parts with ~2.4% and one for the larger ones with 0.8%. They are stored at our desk.
