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Trouble with PCR after cloning

PCR cloning

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#1 Saadlee

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Posted 01 August 2018 - 02:47 AM

Hi,
 
I did transformation and found some colonies in the ampicillin plate having Uncut vector and competent cells . And also in the plate with Insert+vector and competent cells. Then, I picked 5 colonies from insert+vector plate and 1 colonies from uncut vector plates. Boiled DNA template was prepared from 6 of them. Then, I did PCR for 6 of them. My positive control was miniprep vector plasmid DNA. The concentration was 300 ng/ul. During PCR, I used 1 ul of template for 7 reaction. I set up 25 ul reaction. Other components were,
10 mM dNTP = 1 ul, taq polymerase = 0.25 ul, 10uM primer (F and R) = 1 ul each, 10x buffer = 5 ul.
The reaction condition was following:
 
Initial denaturation : 94 for 2 min (1 cycle) , denaturation : 94 for 30 s, Annealing: 55.3 for 30s, elongation: 72 for 45 s ( all three steps for 30 cycles), Final elongation: 72 for 7 min
 
My expected band size was ~300 bp. There is no band for 6 colonies that I picked from transformed plate. But got two bands for the miniprep vector ( one near 5 kb, other 8 kb but faint). The vector is itself 5 Kb. I think PCR didn't work. I expected to have band around 300 bp. Why those band appears? Is it the problem with designing primer or isolation of plasmid? How to troubleshoot it?
 
Cheers


#2 OldCloner

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Posted 01 August 2018 - 07:13 AM

Hi,

 

Where are your primers supposed to stick? Are they on either side of the vector’s cloning site? That would mean you get some tiny little product if the insert is not present, but a ~300 bp product if it is present? How big should the tiny little product be? Or are you using primers that are specific to the inserted band? I’ll assume the former, since you are using only the vector plasmid as a positive PCR control. That implies the primers stick to it (the vector).

 

First, your PCR reactions: Your positive control: it looks like you put 300 ng of plasmid into a 25 ul rxn. Way too much! Way, way too much!  What you see on the gel might just be leftover template you put in there.  Dilute your control down to about 1 ng/ul and use that. Or less! For plasmid, picogram quantities should be sufficient. Also, if your primers are very close together (as I speculated above) the tiny band you would get might be too small to see on your gel.

 

It also appears you are doing colony PCR, boiling some of the colony to lyse it, then putting 1 ul in the PCR rxn. If you are using E. coli you don’t really need to boil first. Put a little bit of colony into a tube with 50 ul dH2O with a sterile toothpick and swirl it a bit. Do not use much of the colony-if the water is very turbid it is too much. Just get a very slight turbidity or even no visible turbidity- trust that it is in there. Use 1 ul as PCR template. If the PCR rxn looks turbid after adding the bacterial template it is too much. I know from personal experience, if you get too much colony into the rxn it is inhibitory, and you get nothing. Anyway, the denaturation at 94 will lyse the bacteria just fine, boiling is just extra work.

 

Now, back to your transformation. There should have been a negative control with ligated cut vector + NO insert. You were supposed to do something to insure that the cut vector would not just re-ligate itself. Usually that is a treatment with alkaline phosphatase (AP) of some sort. Or, maybe you cut the vector with two different restriction enzymes, and purified it to get rid of the tiny little fragment between the two R-sites. Anyway, if things were done right you should have gotten very few colonies on this negative control plate, definitely fewer than the experimental plate (vector + insert). Without this control it is hard to guess whether your colonies on the experimental plate are likely to be background (re-ligated empty vector) or successful insertions, which would be useful to know before you begin screening.  The positive control you described (uncut vector) is useful for telling you your competent cells are good, but it does not tell you more than that.

 

If all or (most) of the colonies on your experimental plate are background, and if there is only a tiny space between the primer binding sites, you might not see anything after PCR, unless you are running a polyacrylamide gel. For PCR, it would be helpful to have a positive control plasmid that already has an insert in it.

 

I also noticed you put in 5 ul of 10 x buffer in a 25 ul rxn.  Isn’t that too much? And by the way, can you do blue-white screening with X-gal?


Edited by OldCloner, 01 August 2018 - 07:16 AM.






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