Day 17: Treating C17 cells with p110 peptide                     

It was required that we diluted the stock solutions which were available to us to make up the desired concentrations for the wells: 0.1μM, 0.3μM, 1μM, 3μM, 10μM.  The TAT and Myr-p110 were both 10mM stock solutions whilst TAT-p110 was 4mM stock solution as it would not dissolve properly previously.
The desired final concentrations meant that we had to dilute the stock solution into 1/10 and 1/100 solutions:


-          Put 45 μl media into each of the tubes – the aim is to have 50 μl total volume by the end of the dilution.
-          Take 5μl TAT Stock 10mM and place it into the 1/10 TAT tube. Vortex to ensure that the stock has dissolved properly.
-          Take 5μl from 1/10 TAT and place it into the 1/100 TAT tube, also vortex to ensure the mixture is properly diluted.
-          Repeat these steps for the Myr-p110 and the TAT-p110.
     

The overall volumes required:


Example alculation of the volumes of each of the diluted peptides required to make up final concentrations 0.1μM, 0.3μM, 1μM, 3μM, 10μM in each well for 500μl media:
Use the equation V = n/C
For TAT and Myr-p110 (Both 10mM stock solutions):
 To make 0.1μM final: Use the 0.1mM dilution tube, ie. 1/100. 0.1mM is the same as 100μM.
Therefore: 0.1 μM/100 μM = 1x10-3.
1x10-3 x 500 μl = 0.5 μl/500 μl media in ONE WELL.
For 5 wells: 0.5 μl / 500 μl x 5 = 2.5 μl/2.5ml media
The treatment of the plates:

-          Aspirate the media off all of the plates and add new the calculated concentrations of treatment to each well in the corresponding concentration columns. On 0, just add 500 μl media to each of the wells.
-          Once complete, note down the time it went in to the incubator.
-          Keep in the incubator for the next day.

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