Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/7 August 2015"
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Revision as of 21:15, 11 August 2015
8/7
- Record the weight of the pellets.
- In total, the pellet weighed 1.75 grams, or about 0.6 grams per 100ml, which is lower than the 0.8 grams we have gotten in the past.
- Transfer pellet to one falcon tube.
- Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing.
- Put on shaker or rotating mixer for 15 min at RT.
- Take first fraction, (F1) of this full cell lysate.
- Centrifuge 16000 g 20 min at 4 degrees C
- Take second fraction of this supernatant (S1) which should contain soluble proteins, while our desired protein should be in the pellet.
- Resuspend in the same volume of 1X Bugbuster 8.75ml as above.
- Make sure the pellet is completely resuspended!
- Take Fraction at this point (F2) Cell lysate #2
- Add DNAse (1 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
- Add dry lysozyme to concentration of 200 ug / ml
- Dissolved the lysozyme in water and added the water.
- Let incubate on ice 30 minutes, swirl every 5 min.
- Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
- Add 6 volumes of 1:10 diluted bugbuster (.1X)
- Can split up into two falcon tubes if necessary.
- Vortex for 1 minute
- Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
- Collect supernatant as fraction (S2). Inclusion body should be the pellet.
- Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
- Used 10 ml of SDS. (Final)
- Store at 4C for further processing and analysis.
