I was doing some cloning, and everything was going swimmingly. I only used a single restriction enzyme for the insertion (I know, I know, so sue me). Anyway, the gene was inserted in the incorrect orientation, with the start codon at the 3' end of the plasmid. Is it possible to somehow finagle it into proper orientation via some kind of miraculous molecular technique, or should i just restart and do it the less lazy way?
When you use a single restriction enzyme, you have a 50-50 chance of getting the fragment cloned in either orientation. Therefore if you isolate 20 or 30 different clones you should end up with a few in the right orientation. One other option is to isolate the fragment with a double digest, clone into another vector and then cut it out using appropriate RE combination to do a directional cloning into the destination vector.
I have never heard of a way to do this. If there is, I would imagine it wouldn't be any easier than just screening more colonies or trying again.
What are the details of your assembly? I would argue there's really no such thing as a 3' end of a circular plasmid anyway. If your gene came as one chunk with a promoter and terminator maybe you can just live with the "backwards" gene?
I had to do a cloning protocol using one restriction enzyme alone, because my plasmid uses only that enzyme. So, to make sure that I had the correct orientation and only one copy of my fragment of interest (because you can also get more that one copies inserted in tandem!) I screened a lot of my transformant colonies till I found the one I needed. I hope you still have your transformation plate so you can go back screening!
I do have my plates. Based on the 50-50 thing mentioned by Sakthikumar, I am planning on doing a double digest of a bunch of colonies and to digest them with two enzymes, one which cuts the 5' end of the plasmid and one which cuts at the 3' end of the fragment. This will identify if any of the colonies have a correctly inserted gene. Unfortunately, I cannot use the backwards gene, it has to be oriented correctly due to the promoter region location in the plasmid and not in the fragment.
Oh and Barbara how did you screen your colonies? We did clone checker followed by sequencing.
I use colony PCR with one primer on the vector and the other one on the fragment, so the presence and size of the band will give you an idea of orientation and if only one or more fragments were inserted. For the positive samples I do a miniprep and sequencing. In my experience, this is the fastest, simple way to screen a lot of colonies at once.
We used a similar approach as Sakthikumar and Barbara. We always check at least 20 clones by a colo-PCR using primers that identify the orientation of the insert, usually half of them have to be in the right direction. Afterwards we perform a control digest (if the plasmid contains appropriate restriction sites in and outside the insert) or sequencing.
If your plates are not too old you can also use alternative restriction digest to screen for correct plasmids. Check your cloning map and identify restriction sites which will give you different restriction products of different size depending on whether your insert is in the correct or wrong orientation. It saves you designing and waiting for primers if you haven't got the ones you need. Good Luck!
It is also possible that your gene stresses the cells or is even lethal.
I guess you use E.coli as recipient, which can also express genes from gram positives and even yeasts to my experience.
To check this, you have to go the more sweating way as our colleaques mentioned before.
"Per aspera ad astra", Good Luck!
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