Team:WashU/Protocols/Celllysis

From 2012.igem.org

(Difference between revisions)
(Created page with "__NOTOC__ <!--Thanks to UCSD for the above instructions. The original source can be found here: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clari...")
 
(3 intermediate revisions not shown)
Line 1: Line 1:
__NOTOC__
__NOTOC__
<!--Thanks to UCSD for the above instructions.  The original source can be found here: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/ under the "General lysis buffer" section. EDTA was substituted for DDT and NaCl was not included as we did not have it currently available at the time of creation-->
<!--Thanks to UCSD for the above instructions.  The original source can be found here: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/ under the "General lysis buffer" section. EDTA was substituted for DDT and NaCl was not included as we did not have it currently available at the time of creation-->
-
{{WashUback}}
+
{{WashUbackprotocols}}
 +
<div id = "protocolshort">
==Cell Lysis and Protein Purification Prep==
==Cell Lysis and Protein Purification Prep==
Line 12: Line 13:
<br>
<br>
Lysis:
Lysis:
-
# The
+
# Spin down the cells desired in a microcentrifuge for 1 minute at 12,000RPM. Discard supernatant and resuspend the culture in the buffer above.
 +
#Sonicate or use some other method to extract proteins.
 +
#Refer back to the SDS-PAGE protocol to see how to load the gel.
 +
 
 +
 
 +
 
 +
<div align="center">
 +
<font size ="5">
 +
[https://2012.igem.org/Team:WashU/Protocols Back to Protocols]

Latest revision as of 21:34, 9 August 2012


Cell Lysis and Protein Purification Prep

Buffer:

  1. The appropriate amount of tris(hydroxymethyl)aminomethane (Tris) was weighed to create a 50 mM solution. (6.057g/ liter of solution).
  2. Similarly, ethylenediaminetetraacetic acid (EDTA) was weighed out to create a 1 mM solution. (0.292g/ liter of solution).
  3. The two substances (Tris and EDTA) were dissolved in deionized water. (The solution had some difficulty dissolving and so was placed in a 37°C water bath for a few hours.)
  4. Now, the solution was pH-ed using 1 M HCl to a pH of 7.5. (NaOH may be added in the event of an overshoot.)


Lysis:

  1. Spin down the cells desired in a microcentrifuge for 1 minute at 12,000RPM. Discard supernatant and resuspend the culture in the buffer above.
  2. Sonicate or use some other method to extract proteins.
  3. Refer back to the SDS-PAGE protocol to see how to load the gel.


Retrieved from "http://2012.igem.org/Team:WashU/Protocols/Celllysis"