As a result, one of the bacteria was contamination, didn't express protein phaC and didnt contain the correct plasmids.
Then I continue to culture and induce the correct bacteria with a total amount of 2 litre media. (5 X 400ml culture)
After last weekend rest, I'm ready to continue to the next step, here is the plan.
17 AUG 2010 (Done)
1. SDS PAGE (for total protein expression)
18 AUG 2010
1. Sonication for 400ml culture (2 X 250ml centrifuge tube culture, others were kept for further uses at -20C) (DONE)
2. SDS PAGE (to check the solubility) (DONE)
3. PhaC temperature stability test. (just choose 2 different temperature, 4C and RT ~25C, I'm wondering the stability of my PhaC protein, so just wanna get more information about its degradation rate.) (Reschedule to 19 AUG, need to use pure protein)
19 AUG 2010
1. Purification by TALON
2. Purification by denatured conditions (TALON as well, doing this to check whether the suspicious 30kDa protein is the complex of PhaC protein or not)
3. SDS PAGE (to check the purity)
20 AUG 2010
1. Gel filtration (optimized condition, slower flow rate, smaller volume sample)
2. SDS PAGE
21/22 AUG 2010
1. PhaC enzyme activity assay (Joanne)
*Progress of the plan flow will be indicated and post will be edited accordingly.
Also as a guideline to push my laziness away.
*At the same time, 20AUG2010 will be our small group meeting for the structure of PhaC review which is 1 week later than our predicted schedule.
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