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Unusual cloning issue... anyone else??

cloning colony PCR DNA Digest

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4 replies to this topic

#1 jillparkin

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Posted 09 August 2013 - 04:31 AM

I have encountered an unusual problem with my cloning and wondering if anyone else has experienced this and what the problem might be.

I have performed a simple cloning using digest/ligation etc. All steps appeared to work fine and I got plenty of colonies from my E.coli transformation. I screened colonies by colony PCR and found some candidates with the expected size product.

However when I went to confirm these candidates by restriction digest I found multiple bands on my agarose gel. At first I presumed a mistake (wrong enzymes perhaps) so I repeated the miniprep and this time before doing the digest I ran a sample of just DNA on a gel.

This also showed multiple DNA bands per sample, so my question is how is this possible when I only transformed 1 DNA ligation product into my competent cells? Could it be some sort of contamination? But it was in every sample (although not the same sizes of band patterns in every one).

Any thoughts/advice/solutions greatly appreciated.

Jill



#2 loz

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Posted 09 August 2013 - 10:27 PM

Hi,

 

I've had a quick think when I have seen such things. I would have thought multiple digestion sites in your vector/insert and suggtested that you check sequences or questioned if the the digest gone to completion or star activity from your enzymes but as I understand it you ran an undigested DNA sample along side your digested one post PCR right?

Assuming you have controls, could it be unspecific binding by PCR primers to your sequence? Secondary primer structures? PCR conditions? Have you tried running just the miniprep before doing any digests along side a digest of the miniprep before any of the PCR? You could also check by digest your vector material before digesting and ligating the material. Could you could also screen your vector/insert products by picking off a few colonies and digesting, then sequence instead of colony PCR? What about a gel purification, cut out the band from agarose gel of the correct size and clean it up with a kit?

 

That is all I can think of for now. Good luck!



#3 Glynn

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Posted 19 August 2013 - 04:38 PM

This has happened to me too! 

Are you sure about the colony PCR?  I've seen false positives with this.  Also like loz said, maybe purify it more, especially gel purification.  Make sure you clean out the gel box really well beforehand too, if it's used frequently there could be residual DNA.  You also could have ligated more than one insert into the vector, especially if the insert is small.  A ligation may produce a mixed population of plasmids, so it's possible that one colony could have a vector with a single insert, while a different colony could contain a plasmid with two or three copies of your insert.  If the insert is large, though, this is unlikely.

I would gel purify and then just go ahead and sequence a few of the clones that looked correct.



#4 phage434

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Posted 20 August 2013 - 09:31 AM

Undigested miniprep DNA will often (usually) show multiple bands, due to supercoiling. This does not indicate several different plasmids present in the preparation.

Multiple unexpected bands in your digested DNA suggests that the sequence of the plasmid you expect is not the one you have produced. Adding th fragment lengths should tell you the length of the parent plasmid (though, watch out for overlapping bands). Finding a single cutting enzyme would tell you the plasmid length. I'd also recommend sequencing the plasmid to really tell you what is happening.



#5 susnata

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Posted 23 August 2013 - 01:18 AM

well.. i might be gravely mistaken.. n i don't know how you did the cloning.

 

but if your insert has formed multimers n then got ligated to the vector, that might explain the multiple bands.

 

in such a case, there might be plasmids with 1 insert or 2 inserts or even many inserts. .. n during transformation a cell could have taken up more than a plasmid. so even if you run the uncut plasmid you may see multiple bands because they will differ in size.

 

if you run a pcr, then each amplicon may of different sizes depending on the number of times the insert is repeated...

 

 

a single digestion of the plasmid isolated from your clones might help you to understand if there are multiple plasmids in the clone.







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