Transformant plating advise - How long can I store my transofrmants at 4'C? (Jul/18/2009 )
Hey everybody!
I did a transformation into my DH5a competent cells yesterday and plated them on my lb/amp/iptg/x-gal plates. But I think I plated too much transformants (100 ul that was pre-incubated) or had too little amp (~100 ug/ml) as I have bacterial lawns on my plates. So I was going to redo my plates, but I was wondering if I could still use my tranformants that I stored at 4'C overnight?
Plus, looking at my plated I can see three types of colonies: two large colonies (blue and white, respectively), and smaller white colonies (looks like satellite colonies...). If you were feeling "adventurous", would you think that the larger white colonies contained the inserts since I had blue ones of the same size?
Thanks for the help!
Hi LordPhantom -- welcome to the BioForums!
Were I you, I would select several of the white and light-blue colonies and streak them to Amp/X-gal plates. If the colonies are too crowded to do that, I would take some of the growth and streak it to Amp/X-gal plates for single colonies.
I think what you're seeing is just the result of plating too heavy a load -- you have some large white colonies (likely what you're looking for), large blue colonies (cells with vector alone in them), and small white colonies (non-transformed cells arising as satellite colonies).
The transformants you held at 4C might still be okay, depending on what media they were in. But, as I said above, you already probably have the transformants you're looking for on your original overloaded plates, so I'd recover from there...
LordPhantom on Jul 18 2009, 01:33 PM said:
I did a transformation into my DH5a competent cells yesterday and plated them on my lb/amp/iptg/x-gal plates. But I think I plated too much transformants (100 ul that was pre-incubated) or had too little amp (~100 ug/ml) as I have bacterial lawns on my plates. So I was going to redo my plates, but I was wondering if I could still use my tranformants that I stored at 4'C overnight?
Plus, looking at my plated I can see three types of colonies: two large colonies (blue and white, respectively), and smaller white colonies (looks like satellite colonies...). If you were feeling "adventurous", would you think that the larger white colonies contained the inserts since I had blue ones of the same size?
Thanks for the help!
I've gone back and plated electroporated E. coli a couple days after storage in 4C, and they grew fine. They were stored in LB media.
Thanks for the quick reply and welcome guys!
The cells were stored in SOC media which was used to pre-cultivate them. I'll try growing them... I'll let you know if I see anything.
I'll also try streaking some of the larger white colonies. Thanks HomeBrew.
LordPhantom on Jul 18 2009, 10:07 PM said:
Not sure what "pre-cultivate" means...
LordPhantom on Jul 18 2009, 10:07 PM said:
Cool.
LordPhantom on Jul 18 2009, 10:07 PM said:
This is your best bet, I think...
LordPhantom on Jul 18 2009, 10:07 PM said:
No problem -- thats's what we're here for. Good luck!
HomeBrew on Jul 18 2009, 07:27 PM said:
LordPhantom on Jul 18 2009, 10:07 PM said:
Not sure what "pre-cultivate" means...
I just meant that I incubate the tranformants at 37'C for 1.5 hours before I plate them.
HomeBrew on Jul 18 2009, 07:27 PM said:
LordPhantom on Jul 18 2009, 10:07 PM said:
Cool.
I actually got a few colonies... but unfortunately they were all blue
Oh, well... you live and learn I guess 
1.5 hour is unneccesary if it's Amp.
0.5 or even direct plating is fine sometimes.
Amp doesn't kill the cell right off like Kan
the cell will be able to still synthesize protein i.e the beta-lactamase prior to dividing
hanming86 on Jul 22 2009, 11:17 AM said:
0.5 or even direct plating is fine sometimes.
Amp doesn't kill the cell right off like Kan
the cell will be able to still synthesize protein i.e the beta-lactamase prior to dividing
I agree. There is no phenotypic lag with amp; all you're doing with a non-selective incubation prior to plating is allowing more of your non-plasmid containing cells to divide.
hanming86 on Jul 22 2009, 10:17 AM said:
0.5 or even direct plating is fine sometimes.
Amp doesn't kill the cell right off like Kan
the cell will be able to still synthesize protein i.e the beta-lactamase prior to dividing
That is very good information of which I was not aware.
HomeBrew on Jul 22 2009, 08:21 AM said:
hanming86 on Jul 22 2009, 11:17 AM said:
0.5 or even direct plating is fine sometimes.
Amp doesn't kill the cell right off like Kan
the cell will be able to still synthesize protein i.e the beta-lactamase prior to dividing
I agree. There is no phenotypic lag with amp; all you're doing with a non-selective incubation prior to plating is allowing more of your non-plasmid containing cells to divide.
that actually makes sense and makes me 1.5 hrs happier! Thanks for the sweet tip!