Team:EPF-Lausanne/Notebook/19 July 2012

From 2012.igem.org

(Difference between revisions)
(→Morning: Cell counting and sampling)
(→Morning: Cell counting and sampling)
Line 5: Line 5:
<!-- Note: a list of all protocols can be found here: -->
<!-- Note: a list of all protocols can be found here: -->
<!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol  -->
<!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol  -->
 +
'''Cell counting'''
 +
Given our cell density we used a 12x dilution (table). We transfered 6 samples of cells into a 96 well plate, added Trypan blue and analysed each sample with Countess.
 +
[table with results]
 +
'''Western Blot sampling'''
 +
Protocol (add to separate page):
 +
#Centrifugation of the cell samples at 2500xg for 10min. Discard the supernatant (vacuum).
 +
#Washing: Resuspend the pellet in PBS1x, centrifuge at 2500xg for 10min, discard supernatant.
 +
#Add at least 1mL PER-M Reagent for 100mg (~100µl) of cell pellet. Pipette up and down to suspend.
 +
#Shake gently for 10min.
 +
#Remove cell bedris by centrifugation: ~1400xg for 15min
 +
#Transfer the supernatant into new tubes for analysis or storage.
 +
 +
*Samples where taken from odd numbered tubes : 1,3,5,7.
 +
*We used 300µl of M-PER Reagent based on the size of our pellets.
 +
*Maximum centrifugation was 10'600xg.
{{:Team:EPF-Lausanne/Template/Protocol|None}}
{{:Team:EPF-Lausanne/Template/Protocol|None}}
; Comments:
; Comments:
 +
For samples 3 and 5 we had to repeat step 5 (centrifugation to remove cell debris) because the cell pellet got aspired in the pipette while trying to transfer the supernantant into fresh tubes.
== Afternoon: Cell lysates ==
== Afternoon: Cell lysates ==

Revision as of 21:47, 19 July 2012

Contents

Morning: Cell counting and sampling

Cell counting Given our cell density we used a 12x dilution (table). We transfered 6 samples of cells into a 96 well plate, added Trypan blue and analysed each sample with Countess.

[table with results] Western Blot sampling

Protocol (add to separate page):

  1. Centrifugation of the cell samples at 2500xg for 10min. Discard the supernatant (vacuum).
  2. Washing: Resuspend the pellet in PBS1x, centrifuge at 2500xg for 10min, discard supernatant.
  3. Add at least 1mL PER-M Reagent for 100mg (~100µl) of cell pellet. Pipette up and down to suspend.
  4. Shake gently for 10min.
  5. Remove cell bedris by centrifugation: ~1400xg for 15min
  6. Transfer the supernatant into new tubes for analysis or storage.
  • Samples where taken from odd numbered tubes : 1,3,5,7.
  • We used 300µl of M-PER Reagent based on the size of our pellets.
  • Maximum centrifugation was 10'600xg.


Protocol: None

Forgot to insert protocol.


Comments

For samples 3 and 5 we had to repeat step 5 (centrifugation to remove cell debris) because the cell pellet got aspired in the pipette while trying to transfer the supernantant into fresh tubes.

Afternoon: Cell lysates

Protocol: None

Forgot to insert protocol.


Comments
Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Notebook/19_July_2012"