< ETHZ(Difference between revisions)
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| - | == Mon, 13. Aug. 2007 ==
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| - | * ''Markus and Martin started labwork at 3pm.''
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| - | * <b>Cutting out the parts from the minipreped DNA ( ) with the restriction enzymes EcoRI and PstI:</b> <br> Prepared 4 ED with numbering 11-14 <br> Poored in each: 0.1 µl BSA, 0.5 µl EcoRI, 0.5 µl PstI, 1.0 µl Buffer, 2.9 µl steriled water <br> Poored in the different EDs: 5 µl of the uncut DNA from #11-14 <br> 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...)
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| - | * <b>Preparing the Agarose Gel for the electrophoresis: </b> <br> Decided to make a 0.8 % Agarose gel. <br> 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. <br> Waited until the solution was handwarm an then poored it in and waited several minutes until the agarose gel became "solid".
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| - | *<b> Preparing the overnight culture of E.coli Top10 cells for the preparation of competent cells on tuesday: </b> <br> Prepared a sterile Falcon tube. <br> Filled in 5ml of LB liquid in the tube. <br>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. <br> Started incubation over night in the shaking incubator.
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| - | * ''Christos and Tim joined''
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| - | *<b> 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: </b> <br> Putted the "solid" gel in the electrophoresis dish and filled it up with 1xTAE until the whole gel was covered. <br> Prepared 10 times (8 for the DNA and 2 for the markers) 3 µl of loading buffer on parafilm. <br> 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). <br> Pipetted the DNA in the gel, with one marker on the left side and one marker on the right side. <br> Started the electrophoresis and left it running for ~30 min. <br> Put the gel in the EtBr for 5 min and then in the Kodak scanner. <br> 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...
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| - | == Tue, 14. Aug. 2007 ==
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| - | '''Morning shift from 9 am:'''
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| - | * Prepared competent cells. <br>The aliquots are stored in the frezer -80°C in the basement, slot 14.
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| - | '''Evening shift from 5 pm:'''
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| - | * Transformation of , they are in the 37°C shaking incubator. <br>The uncut DNA is stored in the freezer of Andy's fridge, the lowest drawer, in a green rack.
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| - | * Made new Liquid LB and LB Agar (they are at the autoclave)
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| - | * Made some Agarose Gel for the Electrophoresis on Wednesday. <br>One for the small Parts with ~2.4% and one for the larger ones with 0.8%. They are stored at our desk.
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