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Plasmid Amplification Issue


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#1 elephant_talk

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Posted 30 October 2014 - 08:26 AM

I've been tasked to amplify five empty plasmids(all amp resistance), and I ran into some issues that are largely due to my own stupidity. This was the first thing I've done in a lab in nearly 5 months, and I forgot to do a number of things which would have made my life easier. I was provided with ~1 uL of DNA for each plasmid I was supposed to amplify and in doing the transformation, I used up all of the plasmids. So I can't exactly retransform.  After incubation, I yielded plates with many small, close together colonies (ALMOST lawn-like) While picking them for inoculation is within the realm of possibility, it will be difficult to do so without also contacting adjacent colonies. All of the colonies on the plate should have the empty plasmids, so perhaps my caution isn't as warranted.  What should I do? 

 

Things I forgot to do:

Save the remaining cells

Plate several dilutions

 

Thanks for the advice.



#2 bob1

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Posted 30 October 2014 - 09:42 AM

What I would do is pick the colonies you want as best you can, streak each as a small line on a gridded plate (so you have a backup), and put the tip into broth. You should then be able to grow a broth that you can use for re-plating for single colonies. You will of course, need to screen for the presence of your plasmid, but at least you should be able to get some single isolates out.



#3 elephant_talk

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Posted 30 October 2014 - 10:12 AM

Thanks for the advice bob1

 

Here's another related solution that I thought of, and I wanted some input. I had some concern about the presence of satellite colonies, but I think those could be weeded out easily enough when inoculating the colonies into LB+amp media. Only those which have the insert should grow in the broth. So my additional selection method would just consist of seeing if the picked colony grew in the broth. I could also streak a small line of each to have a backup, but I wouldn't have to re-plate for single colonies (saving me time).  I don't have to worry about screening for the presence of inserts, because I am just amplifying empty vectors. Is that thought process right? What am I not taking into consideration?

 

Once again, thank you for your input.



#4 bob1

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Posted 30 October 2014 - 11:47 AM

Satellite colonies aren't the problem here - it's mixed populations of plasmids - if you pick more than one colony at the same time, you could (in theory at least) have two or more different plasmids in the mix, so re-plating for single colonies is what I would do. It is also possible that the bacteria have acquired resistance but lost the plasmid, which is why I suggested screening for plasmid (not insert!).



#5 elephant_talk

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Posted 30 October 2014 - 11:52 AM

I forgot to mention that the plasmids are not mixed. I did five separate transformations (one for each plasmid). Are you saying that even within each of these five separate transformations, there could be a mixed population of plasmids? If so, why is that the case? Thanks for the input! You are an awesome resource.


Edited by elephant_talk, 30 October 2014 - 12:11 PM.


#6 bob1

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Posted 31 October 2014 - 12:56 AM

In theory at least, each colony results from a single bacterium which has taken up a single copy of the plasmid. Also in theory, occasionally (more often that you might suppose given the number of replication cycles and number of bacteria) some of those copies will be mutated (often non-consequential), so if you take more than one colony, one of the plasmids you grow up of them might be mutant and cause you problems downstream.



#7 phage434

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Posted 31 October 2014 - 06:39 PM

Easy fix: Use a loop to pick up some of your lawn and restreak for single colonies on a new plate. Pick a single colony, go. Keep the plate until you verify that you have your plasmid.






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