Getting Bacteria from an OD0.8 to an OD 0.1
Posted 15 June 2013 - 07:06 PM
Say I have 50mL of bacteria at an OD0.8 and I need to get bacteria to a reading of 0.1..this is what I've been doing in the past but just want to confirm that what I've been doing in correct.
So from my 50mL, I took 1mL, spun and then resuspended the pellet in 1mL of PBS. (I now have 1mL of bacteria at OD0.8 in PBS)
I took 125uL from this and added to 875uL of PBS (OD has gone down 8x now)
Is this correct--would this now give me 1mL of bacteria at an OD0.1?
Posted 15 June 2013 - 10:24 PM
The reason I ask is that when I am making competent bacteria, it is vital that the OD reading be on the original culture- as this is an indication of growth phase and will affect the quality of my end product cells (their transformability).
So if I did what you did, it would be pointless, as the cells would still have grown to the 0.8 density. If that makes sense?
Posted 15 June 2013 - 10:34 PM
Posted 15 June 2013 - 11:03 PM
In terms of consistency between your experiments- this is probably fine. In that you will be adding the same number of cells each time (regardless of what this number is).
But, and I *should* know this, is the relationship between OD and cell number linear (I have a feeling it isn't?).
Because then the number of cells in your dilution (1/8 of an OD 0.8 culture) won't be the same as the number of cells in 1ml of a culture at OD of 0.1
Posted 16 June 2013 - 12:25 PM
Posted 16 June 2013 - 07:14 PM