LR reaction gateway system

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LR reaction gateway system

Postby kakafonie » Jul 19 2015 3:03 pm

Hello all,

Has anyone here every noticed something weird when doing a LR reaction in terms of getting some sort of mixture of plasmids? Like some sort of hybrid plasmid?

I get a very low efficiency after the LR reaction (only a few cells per plate). The destination vector is fine (all gateway sequences are ok), but after the LR reaction I get only a few colonies and when I sequence them I get something really strange.
I use 3 primers:
1 primer on my destination vector before the GOI (primer 1)
1 primer after my GOI on the destination vector (to cover the gene and some part of my destination vector) (primer 2).
1 primer on the GOI , going towards my destination vector (primer 3).

The first 2 primers give me what I want:
Primer 1: piece of my destination (now expression vector) , attb1 site, followed by the GOI

Primer 2: piece of my destination vector , attb2 site, followed by the GOI

The above primers, "added" together give me: destination vector - GOI - destination vector.
So far so good.

However: using primer 3 (that binds my GOI) I get : GOI followed by a little piece of the attb1 site and then the donor vector...
This makes no sense to me.

Do I have some sort of hybrid plasmid? But how is this possible? Or do I have a mixture of plasmids? But this seems also weird since its from a single colony (I hardly have any colonies on my plate anyway so its really form a single CFU).


I know that something is wrong since I only get a few colonies per LR reaction after plating, but I really do not get this hybrid/weird plasmid.

When putting it on a gel, I just seem to see 1 single band. So its one plasmid? But how is this possible? one plasmid with 3 attb sites?
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Re: LR reaction gateway system

Postby mchlbrmn » Jul 20 2015 4:41 pm

When cloning with ligase you can sometimes get dimers: RE1-Vector-RE(restriction site)1-insert-RE2-Vector(repeated)-RE1-Insert(again)- RE2, 2 of everything all circularized. With any primer the sequence will be normal, since the sequence run can't go all the way around and find that it's all repeated.
I can't understand why your primer 3 comes out with clean but incorrect sequence. If you can see the primer landing site in the sequence data from primers 1 and 2, then you've seen that primer 3 should give a normal sequence at least one time. If you have a weird clone where the insert is (partially or completely?) repeated, then you would get two superimposed sequences. The sequence would start normal in the GOI, and then would become messy overlapping sequence at the point after part of the attB1 where it becomes abnormal in one part of the clone (or on a separate plasmid mixed in), and normal in the part sequenced by primers 1 and 2. Did you look at the actual sequence chromatogram, rather than just the sequence it gives you? Is it possible that there is a fainter underlying second sequence starting at that point in sequence from primer 3 that is overpowered by the odd sequence?
In any case, it's suspicious to start with that you got so few colonies, and a signal that something is wrong. I've also seen weird things in that situation.
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Re: LR reaction gateway system

Postby kakafonie » Jul 21 2015 10:25 am

mchlbrmn wrote:When cloning with ligase you can sometimes get dimers: RE1-Vector-RE(restriction site)1-insert-RE2-Vector(repeated)-RE1-Insert(again)- RE2, 2 of everything all circularized. With any primer the sequence will be normal, since the sequence run can't go all the way around and find that it's all repeated.
I can't understand why your primer 3 comes out with clean but incorrect sequence. If you can see the primer landing site in the sequence data from primers 1 and 2, then you've seen that primer 3 should give a normal sequence at least one time. If you have a weird clone where the insert is (partially or completely?) repeated, then you would get two superimposed sequences. The sequence would start normal in the GOI, and then would become messy overlapping sequence at the point after part of the attB1 where it becomes abnormal in one part of the clone (or on a separate plasmid mixed in), and normal in the part sequenced by primers 1 and 2. Did you look at the actual sequence chromatogram, rather than just the sequence it gives you? Is it possible that there is a fainter underlying second sequence starting at that point in sequence from primer 3 that is overpowered by the odd sequence?
In any case, it's suspicious to start with that you got so few colonies, and a signal that something is wrong. I've also seen weird things in that situation.


1) not sure about this, but you call it a ligation? The LR reaction, I do not think this is a ligation? (I do not think the insert is there multiple times or something like this)
2) I checked the chromatograms (also not an expert on this) that I can access at the moment and I did notice that when I have a "bad" sequence (meaning I get this donor vector in the sequencing results) I do have a very bad chromatogram.
In the few cases where I get a good results using the third primer (I have 2 plasmids that do seem to be ok) I do get a nice chromatogram.


The 2 plasmids that seem to work now, I did retransform these in a new E.coli before sequencing them ... somehow this seem to work to get rid of the "bad" plasmid? Could this be possible?
It seems so weird. I still do not understand whether I have 2 plasmids somehow in 1 cell or its some sort of hybrid plasmid...


To me it seems that the LR reaction somehow did not get completed and giving me a co-integrate plasmid , together with perhaps a plasmid that did work?
I just do not understand how this can happen, 2 plasmids together transformed in E.coli? Or is it just 1 plasmid? But then how come that the primers bound to my expression vector give a nice sequencing results? It means there is a "good" plasmid.

Could it be that the plasmid has 3 attb sites at some point? This seems weird also..

+ the fact that on a gel I only see 1 plasmid makes it even more weird! (and its the expected size)
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Re: LR reaction gateway system

Postby mchlbrmn » Jul 21 2015 4:18 pm

1. I was just giving an example of weird stuff during cloning; i didn't mean that you were doing that.
2. Does the sequence become weird/messy at the point where the two sequences diverge? That would be inside the insert if sequence 3 shows a cut off piece of insert followed by Entry clone sequence, and at the att sequence if seq 3 has a normal end to the insert. so the primer landing in Entry or Dest clones would be normal through the insert, then become two superimposed seqs at the end of the insert.
It would make sense that if you had two different plasmids mixed, the Destination clone primers, seqs 1 and 2, would be clean if those primers don't land in the other (Entry?) plasmid, while the insert primer would be present in both Entry and Destination clone, and give clean seq where the seq is identical in both plasmids.
If you had two plasmid bearing bacteria mixed together, or two plasmids mixed in the same bacteria, then retransforming would likely separate the two and give clean clones. If you had one weird plasmid, then retransforming would only help if some kind of rearrangement occurred during the cloning (and cloning is probably the best time to find weird rearrangements made predominant).

My guess ( guess only) is that you have two plasmids, the desired one, and a rearranged Entry clone that deleted the toxic part (and acquired Ap resistnance from the other plasmid?? Or, maybe a chunk of the Entry clone went into the Destination clone, that included part of the Entry vector flanked on both sides by normal insert? OK... so I don't have one best guess... it's just one of those things...
Do check the final clone carefully...
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Re: LR reaction gateway system

Postby kakafonie » Jul 21 2015 6:44 pm

mchlbrmn wrote:1. I was just giving an example of weird stuff during cloning; i didn't mean that you were doing that.
2. Does the sequence become weird/messy at the point where the two sequences diverge? That would be inside the insert if sequence 3 shows a cut off piece of insert followed by Entry clone sequence, and at the att sequence if seq 3 has a normal end to the insert. so the primer landing in Entry or Dest clones would be normal through the insert, then become two superimposed seqs at the end of the insert.
It would make sense that if you had two different plasmids mixed, the Destination clone primers, seqs 1 and 2, would be clean if those primers don't land in the other (Entry?) plasmid, while the insert primer would be present in both Entry and Destination clone, and give clean seq where the seq is identical in both plasmids.
If you had two plasmid bearing bacteria mixed together, or two plasmids mixed in the same bacteria, then retransforming would likely separate the two and give clean clones. If you had one weird plasmid, then retransforming would only help if some kind of rearrangement occurred during the cloning (and cloning is probably the best time to find weird rearrangements made predominant).

My guess ( guess only) is that you have two plasmids, the desired one, and a rearranged Entry clone that deleted the toxic part (and acquired Ap resistnance from the other plasmid?? Or, maybe a chunk of the Entry clone went into the Destination clone, that included part of the Entry vector flanked on both sides by normal insert? OK... so I don't have one best guess... it's just one of those things...
Do check the final clone carefully...


I also think there are 2 plasmids.

You said: maybe a chunk of the entry clone went into the destination clone, well thats why I think is happening: a LR reaction that is not complete: the insert is there followed by a big part of the Attb1 site, followed by the entry clone.
TO me this looks like an incomplete LR reaction: the gene is there, major part of the Attb1 site and then the entry vector rather than the expression clone. So I think the recombination at the attb2 site worked, but the attb1 site is incomplete.
I only know one part of this of course: I do not have a fourth primer that goes from GOI to the attb2 site... (other direction the primer 3). So not sure whether the attb2 site did recombine. But to me this sounds logical.
So the second plasmid is nothing more then the co-integrate plasmid.






Well I do not have acces to the chromatograms to check it right now, but I'll do that asap.


What I find weird is that I got a very low yield after growing the single colonies in the selection medium. If there was a good plasmid (next to the bad one) I would expect a better growth.
This gives me the idea it might just be 1 bad plasmid , however knowing that after a second transformation it did give a good yield... that also makes no sense.

I find it a weird mystery.

I'll be back when I have checked it again.

I wonder if I should use these plasmids in the end or not. Doing another LR reaction will be coslty (I did it already 4 times, each time same problem: only a few colonies) and I doubt it will work all of a sudden.

I have no clue how to be really really sure I have a good plasmid in the end. I'll sequence it again, with the 3 primers , and put it on a gel, if I see one band, I'll think I have the right plasmid.
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Re: LR reaction gateway system

Postby mchlbrmn » Jul 22 2015 6:19 am

You can do several restriction digests that cut out various size bands from different parts of the plasmid, to confirm the thing.
If it does what you need it to do, and you can tell this, then the exact vector may not matter.
You can also just PCR the insert and ligate it into a vector.
(I don't like making decisions; you can tell).
Something is wrong, that you get so few colonies, and weird results. One of the vectors was screwed up, or maybe the insert has homology to a vector and is trying to do it's own thing (I'm just guessing again).
I use very small reactions, 4 ul, to save $.
Maybe it grows poorly because the entire selection gene is not present, or part of the negative selection is.
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Re: LR reaction gateway system

Postby kakafonie » Jul 22 2015 9:23 am

mchlbrmn wrote:You can do several restriction digests that cut out various size bands from different parts of the plasmid, to confirm the thing.
If it does what you need it to do, and you can tell this, then the exact vector may not matter.
You can also just PCR the insert and ligate it into a vector.
(I don't like making decisions; you can tell).
Something is wrong, that you get so few colonies, and weird results. One of the vectors was screwed up, or maybe the insert has homology to a vector and is trying to do it's own thing (I'm just guessing again).
I use very small reactions, 4 ul, to save $.
Maybe it grows poorly because the entire selection gene is not present, or part of the negative selection is.


Well: I can understand some of the reasoning, but its not just with 1 or 2 plasmids bit with 8 ... I can hardly imagine that all 8 inserts have an homology or something os wrong with the gene(s).

Now: I checked my chromatogram today and I did notice that in some the chromatogram gives a bad result when the donor vectors shows up in the sequencing result.
So GOI : sequencing quality still good, donor vectors pops up: quality bad!
So this is indeed something that tells me I might have a mixture.
However: I did not see this in all the samples. Some showed a good quality... weird!!!
It gets weirder the more I look into it.

The RE: I'll for sure keep this in mind as an option and try this later.


And yes: its weird the LR reaction shows little growth, little colonies. But I would think that if I get a low yield in plasmid DNA after the first mini prep, I would get the same result after a second transformation/miniprep and this is not the case. So I do think there is something ...

Its a weird thing!


About PCR approach: yeah, I can do that for now, its just a few vectors, but I am supposed to to 500 of them... I do not see myself doing that by PCR. haha
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Re: LR reaction gateway system

Postby Kiyu » Apr 27 2017 10:07 pm

Hello guys,
Here I am having the same problem with the LR reaction of gateway system. After LR reaction, when I sequence the Forward and reverse primers of GOI, with the reverse primers i got a perfect sequence for GOI but the Forward one i got the donor vector sequence. How it is possible to see the donor vector after LR reaction? is there any way clean it out? I wonder if you share the tips how you solved such a problem.
Best Regards,
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Re: LR reaction gateway system

Postby kakafonie » Apr 29 2017 12:43 pm

Kiyu wrote:Hello guys,
Here I am having the same problem with the LR reaction of gateway system. After LR reaction, when I sequence the Forward and reverse primers of GOI, with the reverse primers i got a perfect sequence for GOI but the Forward one i got the donor vector sequence. How it is possible to see the donor vector after LR reaction? is there any way clean it out? I wonder if you share the tips how you solved such a problem.
Best Regards,


I have not yet been able to really solve it.
What I do think now is that it is not a hybrid plasmid (I changed my mind on this, I think it is not a hybrid plasmid) but in fact the 2 plasmids being present!
So I think you have both the donor plasmid still present and the recombinant destination plasmid (or expression plasmid now).

I noticed that after a few transformations rounds with my "plasmids" I either end up with just the donor plasmid (and dead cells) or (when being very lucky) the destination/expression plasmid (and this living cells after a re-transformation).
However it is not really a good thing, most often (95%) of the time I just wasted time.

I even tried the following trick: digest the plasmid(s) with a RE that only cuts the donor plasmid and not the destination plasmid and then transfer it into E.coli again, however in 99% of the cases this just "killed" the E.coli (no growth because no or almost no real expression plasmid present).
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Re: LR reaction gateway system

Postby 29yrsExperience » Dec 13 2017 9:03 pm

For anyone still following this thread:
It is not necessarily weird you are getting some “entry” or “donor” vector sequence when you sequence your expression construct. Remember that the LR cloning reaction is intended to produce hybrid att sites, as described in the Gateway Technology User Guide as follows: “The DNA segments flanking the recombination sites are switched, such that after recombination, the att sites are hybrid sequences comprised of sequences donated by each parental vector. For example, attL sites are comprised of sequences from attB and attP sites.”

In the case of LR cloning, you start with attL and attR, and end up with attB in the final viable construct. (As a side product you get attP sites attached to a lethal ccdB gene, so those products should not survive in E. coli.) So depending on where you are looking, you will see some entry vector sequence in your expression construct.

Make sure you are using the proper antibiotic for the destination vector (not the same as the entry vector), and make sure your competent cells (for after the LR reaction) are not the kind that “cure” the effect of the lethal ccdB gene in the destination vector (i.e. do not use XL-10 Gold).

Also, I recommend using the Gateway kit control and see if that works for you. If not, contact Invitrogen (or Thermo, or whoever owns this tech at this moment). Sometimes they do actually have kit lots that are not good, and they will replace them with a new lot if the control does not work.

I’ve done some Gateway LR cloning and I usually get a lot of colonies, and most of them are successful. I screen my expression constructs by restriction digest to insure the proper orientation of the insert. (I have seen one with a extra band which most likely represents the entry vector which accidentally got into the same transformed cell as the expression plasmid. Yes, co-transformation with 2 plasmids is possible, but should be rare).
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