Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/3 July 2015"

 
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Beginning of Cell Lysis
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<u>Beginning of Cell Lysis</u>
  
 
The start-up culture incubated overnight in 1 L of LB is removed from the shaking incubator. An OD check is performed, and reported to be 0.65. The contents from the flask are divided up into 20 Falcon tubes, with each one containing approximately 45 mL of start-up culture in LB. The tubes are centrifuged for 15 minutes at 5,000g at 4 degrees Celsius. The liquid is decanted and the cell pellet is weighed. The cell pellet is then suspended in 1X lysis buffer in a 1 to 3 weight (g) to volume (mL) ratio. The tubes are stored at -80 degrees Celsius.  
 
The start-up culture incubated overnight in 1 L of LB is removed from the shaking incubator. An OD check is performed, and reported to be 0.65. The contents from the flask are divided up into 20 Falcon tubes, with each one containing approximately 45 mL of start-up culture in LB. The tubes are centrifuged for 15 minutes at 5,000g at 4 degrees Celsius. The liquid is decanted and the cell pellet is weighed. The cell pellet is then suspended in 1X lysis buffer in a 1 to 3 weight (g) to volume (mL) ratio. The tubes are stored at -80 degrees Celsius.  
  
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Preparation of 1X Lysis Buffer:
 
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5 mL of 8X Lysis Buffer
 
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35 mL of DI H<sub>2</sub>O
 
  
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NOTE: An error occurred when weighing the cell pellets. The weight of the Falcon tube was not cancelled out. Thus the weights of the cell pellets are highly inflated, resulting in much more lysis buffer added than necessary. This means that the cell pellets are much more diluted. However, this is not a huge issue as the quantities of reagents used in the next steps of lysis can be adjusted accordingly.
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<u>Preparation of 1X Lysis Buffer</u>
 +
 
 +
-5 mL of 8X Lysis Buffer
 +
 
 +
-35 mL of DI H<sub>2</sub>O
 +
 
 +
NOTE: An error occurred when weighing the cell pellets. The weight of the Falcon tube was not cancelled out. Thus the weights of the cell pellets are highly inflated, resulting in much more lysis buffer added than necessary. The cell pellets are much more diluted. However, this is not a huge issue as the quantities of reagents used in the next steps of lysis can be adjusted accordingly.

Latest revision as of 23:06, 9 July 2015

Beginning of Cell Lysis

The start-up culture incubated overnight in 1 L of LB is removed from the shaking incubator. An OD check is performed, and reported to be 0.65. The contents from the flask are divided up into 20 Falcon tubes, with each one containing approximately 45 mL of start-up culture in LB. The tubes are centrifuged for 15 minutes at 5,000g at 4 degrees Celsius. The liquid is decanted and the cell pellet is weighed. The cell pellet is then suspended in 1X lysis buffer in a 1 to 3 weight (g) to volume (mL) ratio. The tubes are stored at -80 degrees Celsius.


Preparation of 1X Lysis Buffer

-5 mL of 8X Lysis Buffer

-35 mL of DI H2O

NOTE: An error occurred when weighing the cell pellets. The weight of the Falcon tube was not cancelled out. Thus the weights of the cell pellets are highly inflated, resulting in much more lysis buffer added than necessary. The cell pellets are much more diluted. However, this is not a huge issue as the quantities of reagents used in the next steps of lysis can be adjusted accordingly.