Resuspending bacterial pellet to a certain OD600

[biology_06er]

Hi there,

I plan to do some flow next week and an one of the steps is to resuspend the bacterial pellet to an OD600 0.5. So say I had a 1.5mL overnight culture and the following morning the OD600 was 1.7. Once I spin down, wash etc etc I read to get to a certain OD (0.5 in my case)...this is the calculation


Divide the OD you want (0.5) by the OD bacteria was at in morning (1.7)=0.5/1.7=0.29=giving you a concentration factor (cf)

Divide the original culture volume (1.5mL) by the cf

1.5/0.29=5.1mL


Ok..so I don't need 5.1mL of culture to subsequently use..say altogether I only need 2mL how do I go about this? So that in the end I have 2mL of bacteria with an OD of 0.5?


Man, I hope I make sense!

Cheers,

b_06er

[JackBean]

What about to get only aliquot of your O/N culture?

[biology_06er]

As in from my o.n culture take 0.5/1.7=0.29*2mL=0.588..So do I take 588uL of my overnight culture..spin it down wash etc etc..then resuspend the pellet in 2mL?

[JackBean]

something like that

[biology_06er]

Something like that?..Which means a part is wrong?

[biology_06er]

That sounds wrong..

Is this way better..say I had the a 10mL of o/n starter culture
I take 1mL from that starter culture, wash, spin, block etc..and when it comes down to resuspending my pellet..i resuspend in 1.5mL again..therefore I technically have a culture at an OD600 of 1.7.

From that culture which I just resuspended in...can I take 441uL and then top up 2mL using 1559uL of my buffer?

[JackBean]

If you take 1 ml and dilute it into 1.5 ml, you will hardly have the same OD

[biology_06er]

Urg..I meant resuspened in 1mL

[biology_06er]

and from that 1mL resuspended culture..I take

0.5*2mL/1.7=588uL and top up to 2mL