IGEM:Cambridge/2008/Notebook/Voltage/2008/08/12

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Current revision (09:20, 14 August 2008) (view source)
 
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==Make competent stocks==
==Make competent stocks==
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Chemically competent TOP10 cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C.
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Chemically competent TOP10 cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C.  
-
200ml were spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl<sup>2</sup>, spun down again and resuspended in 4ml CaCl<sup>2</sup>.
+
200ml were spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl<sub>2</sub>, spun down again and resuspended in 4ml CaCl<sub>2</sub>.
 +
Again they were spun down (6500 rpm for 5 mins)and resuspended in 4ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen.
 +
 
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Electroporation competent TOP10 and DH5α cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C.
 +
100ml were spun down at 3800rpm for 8 mins, resuspended in 25ml SDW, spun down again and resuspended in 2ml SDW.
 +
Again they were spun down (6500 rpm for 5 mins)and resuspended in 2ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen.
 +
However lost half of the DH5α when centrifuge tube broke.
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KDP

  • Again try ligation of Kdp and Vector backbone

OsmY

  • Again try ligation of OsmY and B0030 backbone
  • This time try using Xba for B0030 and ligate. Gel again and cut out linear fragment. Cut with Spe and gel - cut out circular fragment.

Make competent stocks

Chemically competent TOP10 cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 200ml were spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl2, spun down again and resuspended in 4ml CaCl2. Again they were spun down (6500 rpm for 5 mins)and resuspended in 4ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen.

Electroporation competent TOP10 and DH5α cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 100ml were spun down at 3800rpm for 8 mins, resuspended in 25ml SDW, spun down again and resuspended in 2ml SDW. Again they were spun down (6500 rpm for 5 mins)and resuspended in 2ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. However lost half of the DH5α when centrifuge tube broke.



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