Competant cells & incompetant scientist - No colonies formed (Sep/25/2006 )
Hi all,
I am simply trying to make a larger amount of a plasmid that I have. I used OneShot TOP10 Chemically Competant cells from Invitrogen with a 6700bp plasmid.
Briefly, what I have done is thawed the cells on ice, added 2ul of plasmid (0.2ug/ml), gently mixed, waited 5 minutes and plated onto ampicillin LB agar plates (100ug/ml) as per instructions that came with the cells. Then left the plates for 16 hours overnight, but no colonies have grown on the plates.
Admittedly I did use the 'rapid' transformation method rather than using the more involved procedure using 42 degree water baths, but I didn't think this would be a problem.
Any ideas as to where I have gone wrong?
davo, did you perform transformation of the control plasmid that came with the kit? this would tell you a great deal
I am guessing you used the appropriate amount of cells, as per directions?
The cells come in 50ul vials of ready to go competant cells that were all to be spread over the plate. There were 10 vials of cells in the pack and I need to clone 6 plasmids and thought that if something went wrong then I'd have more spare vials to use, so I didn't use the control plasmid. I now have 9 vials and still 6 plasmids, or 7 if I include the control. I guess I could use the control but that would be making a fine margin for error!
Do you think 5 mins is a sufficient amount of time for the plasmid to get into the cells before plating? The rapid method is 5 minutes on ice, whilst the regular method is 30 minutes on ice, 30 seconds in 42 degree water bath, add 250ul of SOC medium, shake for 60 minutes at 37 degrees and then spread 20-200ul on a plate and incubate. Perhaps if I did it this way I could use a lower conc of ampicillin and see how that affects it, or even plate out the entire 300ul onto a single plate?
I'm new to this and I am only stabbing in the dark as to the reason for failure!
I would give some time for the cells to recover and start beta-lactamase production before dumping them on selection plate. (I use 30mins)
100ug/ml sounds rather high (although it is within normal range which people use). I perfer to stick to 25ug/ml, maybe even going as high as 50ug/ml. 100ug/ml just sounds excessive.
I have used the same cells, but not the rapid protocol.
The protocol I use requires a 30 min incubation of cells and DNA, a 30 sec heat shock at 42 degrees C, and 1 hr at 37 degrees C prior to plating. I would spend the time and do the full protocol rather than possibly having it not work again.
Using this protocol to simply amplify a plasmid should require only 1 pg of DNA, however if you think your DNA is degraded increase this to 1 ng.
Also, I use only half the cells (25 ul) for each transformation, so you can save on cells that way (add only 125 ul of SOC).
I divide the final solution into 3 plates in case I end up with a lot of colonies. I end up with 150 ul of solution after adding SOC and use 10 ul, 50 ul and 140 ul per plate.
Thanks for your help everyone!
Ucsdgrad88 - what concentration of antibiotic do you use? Is it ampicillin?
I will re-try it using the regular method, and also knock back the concentration of ampicillin on the plates.
Thanks again, I'll let you know how it goes.
Dave
Hello everybody,
I have repeated my transformation using the standard method and lowered the ampicillin to 50ug/ml and it worked! I only got four colonies, I was hoping for more but somebody said four is good and I agree it is better than none!
So I thank you for you help and fingers crossed I can get my other five plasmids to work smoothly.
Cheers
David
Hello People,
I had a look at my plate from last week which grew 4 colonies overnight (of transformed bacteria), and there was several large colonies which had formed over the weekend. Are these suitable to use for plasmid isolation or is it too long after plating? Is there a time limit after plating as to when you should no longer use them for plasmid isolation?
I had a look at my plate from last week which grew 4 colonies overnight (of transformed bacteria), and there was several large colonies which had formed over the weekend. Are these suitable to use for plasmid isolation or is it too long after plating? Is there a time limit after plating as to when you should no longer use them for plasmid isolation?
Yes, there is a limit. Once the antibiotics around the colony has dropped below a treshold value, the cells rapidly lose the plasmid. B-lactamase is secreted into the media, so there is enzyme tends to build up. Amp also gets hydrolysed naturally and degradation rates increases with temperature.
Since you already have proper colonies in hand, I wouldn't use those colonies that appeared on the old plate.