Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/26 May 2015"
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**Based protocol off of the Novagen manufacturer's protocol which can be found [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf here]. | **Based protocol off of the Novagen manufacturer's protocol which can be found [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf here]. | ||
*Expressing honey bee silk in E. Coli, from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001] | *Expressing honey bee silk in E. Coli, from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001] | ||
− | **See [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/18_May_2015 5/18 and [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/19_May_2015 5/19] for details on the protein expression. | + | **See [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/18_May_2015 5/18] and [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/19_May_2015 5/19] for details on the protein expression. |
===Step by Step Protocol=== | ===Step by Step Protocol=== | ||
+ | #Determine wet wet weight of cell pellet after spinning liquid culter at 10000 x g for 10 min. | ||
+ | ## 2.5 g | ||
+ | #Resuspend in 5 ml/g Bug Buster (1x) by pipetting and gently vortexing. | ||
+ | ##This is 12.5 ml in our case | ||
+ | #Put on shaker or rotating mixer for 15 min at RT | ||
+ | ##Took our first fraction at this point of the full cell lysate (F1) | ||
+ | #Centrifuge 16000 g 20 min at 4 degrees C | ||
+ | ##Took next fraction at this point of the supernatant, labeled (S1) | ||
+ | #Resuspend pellet in same volume of 1X bugbuster as before | ||
+ | ##12.5 ml | ||
+ | #Pipett up and down and vortex gently to get an even suspension. | ||
+ | ##Took third fraction at this point (F2) | ||
+ | ##I did not go to great lengths to get an even suspension here, but I noticed in retrospect that the protocol emphasized the importance of this in order to get pure inclusion bodies. | ||
+ | #Add dry lysozyme to final concentration of 200 ug / ml | ||
+ | ##For 12.5 ml solution I added around 2.5 mg of lysozyme | ||
+ | ##'''I did not add any DNAse because it was not mentioned on the manufacturer's protocol, but in the future I will try it with 1 ul of DNAse because chromosomal DNA might have been a problem in subsequent steps. ''' |
Revision as of 19:38, 29 May 2015
BugBuster Protocol
- Using the Bugbuster 10X cell lysis reagent in order to purify our honeybee silk protein via inclusion bodies.
- Based protocol off of the Novagen manufacturer's protocol which can be found [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf here].
- Expressing honey bee silk in E. Coli, from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
Step by Step Protocol
- Determine wet wet weight of cell pellet after spinning liquid culter at 10000 x g for 10 min.
- 2.5 g
- Resuspend in 5 ml/g Bug Buster (1x) by pipetting and gently vortexing.
- This is 12.5 ml in our case
- Put on shaker or rotating mixer for 15 min at RT
- Took our first fraction at this point of the full cell lysate (F1)
- Centrifuge 16000 g 20 min at 4 degrees C
- Took next fraction at this point of the supernatant, labeled (S1)
- Resuspend pellet in same volume of 1X bugbuster as before
- 12.5 ml
- Pipett up and down and vortex gently to get an even suspension.
- Took third fraction at this point (F2)
- I did not go to great lengths to get an even suspension here, but I noticed in retrospect that the protocol emphasized the importance of this in order to get pure inclusion bodies.
- Add dry lysozyme to final concentration of 200 ug / ml
- For 12.5 ml solution I added around 2.5 mg of lysozyme
- I did not add any DNAse because it was not mentioned on the manufacturer's protocol, but in the future I will try it with 1 ul of DNAse because chromosomal DNA might have been a problem in subsequent steps.