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Revision as of 14:05, 2 August 2011

Chemotaxis Lab Protocols

27th of July

The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. This could have been caused by:
-No innoculation
-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.

New protocol:
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.

28th of July

Transformation of cells with 6, 7 and 8:

-Let competent cell strain 5α thaw for around 10 minutes on ice.
-Add 2-3μl of DNA.
-Leave on ice for 20-25 minutes.
-Heat shock cells at 42°C for 45 seconds.
-Leave on ice for 10 minutes.
-Add 500μl of LB broth.
-Incubate for 1 hour at 37°C.
-Centrifuge for 1 minute.
-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. -Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
-Add the rest of the sample to a second chloramphenicol agar plate.


To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:
-10g tryptone
-1000ml of 1X PBS
-autoclave
-1.14g kanamycin

To make 1X PBS (phosphate buffer saline):
-in 800ml of distilled H₂O
-8g of NaCl
-0.2g of KCl
-1.44g of Na₂HPO₄
-0.24g of KH₂PO₄
-adjust pH to 7.4
-adjust volume 1L with additional distilled H₂O
-autoclave
Note: also possibility to use 1X PBS tablets (one tablet per 200ml)