inoculating liquid media from frozen cell stock ok? - (Nov/14/2008 )
I'm reading the protocols for making competent cells, and they all say to streak a plate first and then pick a colony to inoculate an overnight culture before you inoculate that liquid culture into a bigger one for your final prep. my question is: why can't you just go directly from the frozen glycerol bacterial stock directly to inoculating the overnight liquid LB media?
I'm trying to make BL21DE3pLysS cells competent and have chloramphenicol in the overnight LB media.
Thanks!
I'm trying to make BL21DE3pLysS cells competent and have chloramphenicol in the overnight LB media.
Thanks!
By streaking first, you make sure that you are using a single bacterial cell to begin with.
By what you are proposing, if there was a contamination (mixture of bacteria) in the stock, you will end up with a mixed colony of bacteria. Not good.
Since you generate the frozen stock from a clonal population, there is no need to fret over contamination unless you are a slob. Grow your culture directly from your frozen stock. I do it all the time and my plasmid preps generate high yield DNA that sequences perfectly and functions perfectly in several assays. Don't waste your time growing a fresh plate each time unless you are troubleshooting.
Less time = more data = higher chance for "good" data = publications!
When you're making competent cells that don't have any selection on them to start with, so your odds of introducing some contamination are greater if you are not picking a single colony. The cells will probably also be healthier, which will give you better competent cells in the end.
thank you all very much for your input.
i can understand the "healthy" issue, but regarding the potential contamination-- there's chloramphenicol in the liquid media just like there would be on the plate for selection, so why does that make a difference? is a plate somehow more selective? also, if we were just plating this out onto plain LB without any antibiotics (say for making competent DH5alpha cells), wouldn't you have equal likelihood of picking a contaminant colony? maybe i'm still missing a crucial link 
appreciate it!
i can understand the "healthy" issue, but regarding the potential contamination-- there's chloramphenicol in the liquid media just like there would be on the plate for selection, so why does that make a difference? is a plate somehow more selective? also, if we were just plating this out onto plain LB without any antibiotics (say for making competent DH5alpha cells), wouldn't you have equal likelihood of picking a contaminant colony? maybe i'm still missing a crucial link
appreciate it!
1. THe contaminating strain may also have chloramphenicol resistance - after all we all work in labs.
2. The contamination in a frozen stock is always very little, so your likelyhood of picking up a single colony that is wrong out of 1000 or 10,000 right ones is low.
3. Even if you did pick up wrong colony, you being like me:-) you would immediately know when things don't work or start going crazy. With a mixture, you woudn't even know for six months what went wrong. And think of the person you donate your valualbe strain to! She would faithfully waste 2 years of her PhD.
4. There is a value in doing clean expts - the value that you only recognize when you retract your 3 Nature papers.
point taken
thanks!