Question: (Closed) Weird sequencing results after a LR reaction using the gatewaysystem
0
gravatar for Fman
5.1 years ago by
Fman0
Belgium
Fman0 wrote:

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 or multiple plasmids in 1 "cel" ?

I get a very low efficiency after the LR reaction (only a few cells per plate). Which is already a sign something is off.

The destination vector/donor vector are fine, 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) going through this GOI, the attb2 site and further on.
1 primer after my GOI on the destination vector going in the opposite direction (primer 2).
1 primer on the GOI , going towards my destination vector , going to the attb1 site (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 and the attb2 site and my vector.

Primer 2: piece of my destination vector , attb2 site, followed by the GOI and the attb1 site.

The above primers, "added" together give me: destination vector - attb1 - GOI - attb2 - destination vector.
So far so good.

However: using primer 3 (that binds in 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.

A note: when checking the quality of the sequenced results from the primer that binds my GOI (primer 3) I do see that the peaks are not good when I reach the attb1 site (when it goes from GOI to the donor vector rather than expression vector).

So I am guessing I have 2 plasmids rather than 1.

Anyone an insight in this?

sequencing gateway • 1.1k views
ADD COMMENTlink written 5.1 years ago by Fman0

Hello Fman!

We believe that this post does not fit the main topic of this site.

This is not a bioinformatics question, but a wet-lab question.

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.

Cheers!

ADD REPLYlink written 5.1 years ago by Michael Dondrup47k

 

Hello,

I can understand you reasoning, but the reason why I asked here was to get a more detailed answer on the sequencing conundrum.

I wonder if people here ever had this when sequencing it.

But perhaps you are right.

Cheers,

 

 

ADD REPLYlink written 5.1 years ago by Fman0
1

Did you try http://seqanswers.com/ ?

ADD REPLYlink written 5.1 years ago by Michael Dondrup47k
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