biology_06er

Ohh, was the 3rd component the secreted part..do you think its to see if the protein has been secreted. In this case, would the TCA precipitation come from the supernatant in step number 3 (after pelleting my OD 0.6 culture?)

Oook..I'll ask--I was hopoing to have it sorted this weekend..so I could just go in on Monday and start...anyhow..I assume I can use an o/n culture because the protein in constitutively expressed..hence it isn't really a problem if I use the bacteria at SP.

Oh, and trust me I have looked at the lit..but they only state "bacteria was resuspended to an OD of....." that doesn't tell me HOW to do get the certain OD

Hye there,


I am using the same protocol..I just don't get how they did the dilution factor---and I don't want to ask because a) I made it look as though I knew what they were talking about but now I'm not so sure. I can't take the 588 and top up to 2mL because I need to block before hand. I know they told me to take only a little bit of my overnight culture and spin that down as my first step...so the first time I did this experiement this is what I did:



I had a 15mL o/n culture.....the next day I measured OD600 it was 1.7. I took 1.5mL of that culture..I put in eppendorf tube, spun, down, washed, blocked, washed.
The final pellet I resuspended in 1.5mL of buffer. I needed a final sample of 1.2mL at OD600 0.5
From that 1.5mL I took 0.294uL (0.5/1.7) and topped up to 1.2mL

When I started my flow I def had cells...I was told it didn't matter if the OD was not exact but even so, I am sure what I did was wrong above! (well mainly the step in bold)



EDIT==maybe I should have taken 0.5/1.7*(1.2mL)=353uL instead?

Hey Pito!

Thank you so much for your reply..you're a lifesaver!..was stressing out incase Monday came and I went into the lab not 100% sure..and can't really afford to screw it up!..so double checking..I take 588uL out, spin it down, wash, block, wash, and the final pellet I get I resuspend in 2mL?

Um can I let you know the answers to those questions after I've done the exp haha..I'm spending this weekend reading up etc..I'm trying to detect surface protein and I think 0.5 was the ideal OD someone else got when they did a similar exp.

Did you mean you're not a fan of the work I'm doing..such as washing etc? How come?

Thank you again *10000000