Check the ligation product, but got two fragments with strange sizes on the gel - (Jun/15/2008 )

I want to introduce the polyA site from pSTEC into pLoren.
First, I have to cut out the neoR site (~930bp) from pLoren. So, I used double digestion for 2.5 hours (BamHI and NotI), and ran the gel. I got two bands, one is around 10kb, and the other is between 8kb and 9kb. Then I eluted the 10kb band from the gel to get the pLoren without neoR fragment.

Second, I cut out the polyA site from pSTEC using the same double digestion for 2.5 hours (BamHI and NotI), ran the gel. I got two bands, one is around 3kb, and the other is between 200 and 300bp. So, I eluted the 200bp band from the gel, which should be the polyA fragment.

Then, I put the pLoren w/o neoR fragment and polyA fragment together, and used Quick Ligase to ligate these two fragments. After transformed to E-coli, I got a lot of colonies on the plate. I picked several colonies and did the miniprep.
After miniprepration, I check these vectors with double digestion again for 2.5 hours (BamHI and NotI). If the ligation worked, I should get two bands, one is around 10kb, and the other should be around 245bp.

However, from these miniprep (16 tubes), I only got two kinds of results on the gel, which are both strange.

First one is only one clear band between 10kb and 20kb. It could not be uncut vectors. If it’s the cut vector, where is the polyA fragment?? Is that possible that the vector was cut only once? I mean the double digestion didn’t work completely.

The second one is two clear bands, the upper band is between 3kb and 4kb, and the lower band is between 200 and 300bp. If the lower band is polyA fragment, what should be the upper band? Why there is 6kb missing on this vector??

Since I ligated two fragments, there should be no colony if the ligation didn’t work, right? And the large fragment from pLoren should not relegate itself because the two ends are not compatible. It is possible that I included some uncut pLoren in the fragment, that’s why I got so many colonies. However, even if these colonies were from uncut vector, the sizes of the bands after digestion were not correct. So, what would be the problem?

I am a rookie in cloning. Can anyone help me to solve this mystery?? Thanks a million.

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Hi ah do.
Did you load your undigested recombinant plasmid(pLorenpolyA) after plasmic extraction from E.coli as your control? Was the band size correct?

*For your information, i am currently facing this problem too. So i only can speak out my opinion, not solution

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oh, i didn't load the uncut vector. Is it necessary? if it's uncut, how can you decide the size?

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Surely you need the 8-9 kb fragment, because that won't have the NeoR gene...


I don't think you have actually cloned anything. The 10 kb fragment still has the NeoR gene in it. The fact that your miniprep generated DNA between 10 and 20 kb just shows you have supercoiled plasmid. Try growing the cells in Neo and see what you get; any colonies mean you have NeoR in the plasmid.
Thinking about the gel results, do you mean that in one lane you get the 10-20 kb, the 3-4 kb and the 200-300 pb bands? Can you show us a gel image? It will help us a great deal.
Thinking about your second posting about what to run on the gel, you should load both uncut (so you can see the level of supercoiling) and single-cut plasmid (that is, single digest with each of the enzymes). If only one digest works, you know where the problem lies, and you see the linear molecule.

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I don't need the neoR gene, because there is another AmpR gene on pLoren. So, i only eluted the upper band.
From all the colonies i picked, i only got two kinds of results. ( i used 1kb plus ladder)

One was only one clear band (ex. lane 1, 4, and the last one) between 10kb and 20kb. It didn't look like an uncut vector. Rather, it more like the new vector but only been cut once (linearized).

Second was two clear bands (ex. lane 2, 6, and 8). One was between 3kb and 4kb. The other was between 200bp and 300bp. I think this looked like the pSTEC-1 vector, because the sizes matched.
The question is, if i eluted the polyA site from the gel. How come there was still the uncut pSTEC-1 in it? The size of polyA is much smaller the the uncut vector and the rest of the cut vector.

[attachment=4831:scan0001.jpg]

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Another thing is, if i still have the neoR in my vector, then there should be two bands on the gel after digested with BamHI and NotI. I should have got the neoR band at around 1kb, and the other around 10kb. right?

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Unless one of your enzymes is dead...
You will need to do the single digests.

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Why do you think it doesn't have the NeoR gene? What is the size of the plasmid before digestion?
Can you please label the gel? And can we have a labelled image of the initial digestion of your vectors, so we can make comment on it?

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