I am currently doing restriction cloning to clone an insert (size about 4kb) into a vector with size about 3kb. I used two different REs for this cloning, and after transformation, I did colony PCR and got some positive clones with the expected band size.
However, when I extracted out the plasmids from these positive clones and then verified them with different REs, I found that their sizes were totally different from the original one. The plasmid size is about 9kb. Why is it like that? Does anyone know about this, or encountered this situation before?
Among the positive clones, I tested with the REs I used to do the cloning. They give the same number of bands with different band sizes (sizes are different from original insert and vector), as shown in the attached image.
First of all, I will trust the digestion more than the colony PCR. Are you sure that colony PCR was positive? Did you actually amplify the 4kb during colony PCR?
Please check that the bacteria you are using don't have an additional plasmid, by cultivating your transformed bacteria and the empty bacteria side by side and extracting DNA from both using the same protocol.
9 Kb vs 7Kb, the difference is so huge, and therefore can not be your recombinant plasmid. However, check that you are you using the right marker.
Do not forget to run insert and linearized plasmid as additional control in case the marker is not accurate. In a 1% Agorose gel you may not make a difference between 4kb and 4.2 Kb.
So check the basics first and let us know what get.
Hi Antonio,
When looking at your gel picture, I assume that the empty digested vector (3kb) is in the outer left lane of the gel. Are you very sure that this vector is cut? I would not expect to see three bands and surely not one of them being far larger. My opinion is that you are looking at a circular vector which can exist in three different forms and therefore appear at three different sizes when analyzed on an agarose gel....I have seen this happen many times before.
It might very well be that this is also the case for your positive clone.....i.e. that you do have the correct insert (since migration is definitely different compared to the empty vector), but that the control restriction digestion for whatever reason does not work and doesn't cut your DNA.
Hope this helps.
Hi Antonio,
Please recheck your insert seq, there can be a possibility that your insert is having RE site, which you have used for cloning and clone confirmation. May be that's you are getting bands of different size.
Hi there,
Please name the vector that you are using, the restriction enzymes that you are using and I might be able to help you on that. Since just by saying a vector and an insert, audience might not be able to help you around.
egards,
GCK
Most of the suggestions have already been covered here. I can tell you from my failed cloning experiments that even I see such strange plasmids. The expected construct is about 15kb in size and I see something really different, infact bigger or larger than 15kb. My boss tells me that since the empty vector (in my case is big, around14.5kb), there is lot of recombination inside the bacteria, some fragments even get kicked out, because the bacteria wants less workload for multiplying (I guess). He even suspected that some empty vectors might have ligated , concatenating with each other. I know this sounds weird, but this was the explanation we could come up with for the strange plasmids after ligation. I think you might have encountered something similar. In any case colony PCR is never accurate, or at least as accurate as diagnostic digestions after mini preps. I would advise you to repeat your experiments once more, check your ligation products on gel once, before transforming them.
All the best.
If you got larger band than your target band in the gel, this may indicate The restriction enzymes is bound to the substrate DNA, you may need to use Lower number of enzyme units or add SDS (0.1 – 0.5%) to the loading buffer to dissociate the enzyme from the DNA
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