"

From 2012.igem.org

Contents 1 Morning: Cell counting and sampling 1.1 Protocol: None 2 Afternoon: Cell lysates 2.1 Protocol: None

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.

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

Comments