User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/04: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry I</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry I</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==Purpose== | ==Purpose== | ||
* Grow and amplify ADA transfected BL21(DE3) E.coli cells in LB media | * Grow and amplify ADA transfected BL21(DE3) E.coli cells in LB media | ||
* Extract cells from media after ADA protein expression | * Extract cells from media after ADA protein expression | ||
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Settings: Photometric | Settings: Photometric | ||
Data display: Absorbance | Data display: Absorbance | ||
* Centrifuge was turned out to pre-cool to 4°C. | |||
*The absorbance of cells in four Fernbach flasks were as follows: | *The absorbance of cells in four Fernbach flasks were as follows: | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Flask''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume of cell culture[mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Wavelength[nm]''' | |||
| align="center" style="background:#f0f0f0;"|'''Absorbance''' | |||
|- | |||
| 1||2||600||0.683 | |||
|- | |||
| 2||2||600||0.691 | |||
|- | |||
| 3||2||600||0.657 | |||
|- | |||
| 4||2||600||0.701 | |||
|- | |||
|} | |||
* Cell culture were taken out of orbital shaker and transferred into centrifuge bottles. Mass of centrifuge bottles were balanced out to make sure successful centrifugation. The cell culture were placed into centrifuge pre-cooled to 4°C and spin for 30 minutes, 4500rpm, at 4°C. | |||
* Supernatants from centrifuged products were discarded. All four pellets from four centrifuge bottles were resuspended in 30mL binding buffer made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/26|09/26/2012]] with the following condition: | |||
20mM Tris buffer | |||
0.5M NaCl | |||
30mM Imidizole | |||
pH 7.5 | |||
* Cells from pellets were resuspended in the following way: 30mL of binding buffer was first placed in first centrifuge bottle with pellet, cells resuspended using pipetman and vortexer. After cells were completely resuspended in first bottle, the culture were transfered into second centrifuge bottle. This process was repeated until culture containing binding buffer and suspended cell pellets from first, second, third, and fourth centrifuge bottle were well mixed. | |||
* Pellet with binding buffer resuspension were stored in a 50mL falcon tube, and stored in freezer at -80°C. | |||
==Notes== | |||
* IPTG was added to induce cell expression. IPTG was added immediately before cells were placed in orbital shaker to make sure that cells were under ideal conditions when expression proteins. | |||
* The absorbance for cells after 4 hours of growth were higher than expected (an absorbance of 0.600 at 600nm). This indicates the cells were overgrown. The cells were harvested nonetheless. The high absorbance indicates more cells in flasks, thus more protein when purified. | |||
* The cell cultures were centrifuged for 30 minutes as oppose to the protocol which suggests 15 minutes. Longer centrifugation time is used here to make sure all cells were in the pellet. | |||
* The final volume of cell resuspension is 42mL due to the addition of cell pellet as well as small amounts of supernatant that were not completely cleared out. | |||
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__NOTOC__ | __NOTOC__ |
Latest revision as of 22:11, 26 September 2017
Experimental Biological Chemistry I | Main project page Previous entry Next entry | ||||||||||||||||||||
Purpose
Procedure
Molecular weight: 238.31g/mol 0.4M × 0.004L = 0.0016mol of IPTG needed 238.31g/mol × 0.0016mol = 0.3813g of IPTG needed
Wavelength: 600nm Settings: Photometric Data display: Absorbance
20mM Tris buffer 0.5M NaCl 30mM Imidizole pH 7.5
Notes
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