I.e. you have only a single restriction enzyme site available and you want to insert something in a specific direction, BUT you cannot screen for correctly inserted variants later, so you need to insert it in the correct orientation in the first place.
In this case, I am working with a plasmid library, so I cannot use the standard single colony picking and screening method. I also cannot use more RE sites since none are available. Any thoughts?
The short answer in practical terms is going to be no.
I think you should probably rethink how you're going to do this, maybe consider one of the many cloning methods besides traditional restriction enzyme cloning. To give any specific suggestions I'd have to know more about the sequences you're working with and what you're trying to do with them.
Yes you can by using a re enzyme that has a non-symmetrical site like sfiI. (Use the enzyme that is currently available in your plasmid to ligate in two asymmetrical sfiI sites separated by around 25bp which can be easily synthesized as an adapter using primers). You can check this kit.
http://www.takara.co.kr/file/manual/pdf/PT3000-1[1].pdf
Hanna Alalam Thanks for the suggestion - but I don't think it's going to work. While sfiI sites are asymmetrical, when I ligate them in, they will still insert in random orientation, so after digestion with sfiI you still end up with a mix of forward and reverse ligated inserts. No?
Michael Florea actually yes it is directional (the middle part of the restriction site is constructed in a way that the 5' start codon gets cloned in directionally), however, could you tell me what kind library are you working with? is it a cDNA library? are you ligating that you get active expression downstream of a promoter? is your organism eukaryotic? if you would like to take a look at the references yourself the link is below:
Article Directional cloning of cDNA using a selectable SfiI cassette
Method utilizing the sfiI cassette has been patented you can take look at that as well:
https://patents.google.com/patent/EP0773294A2/en
It will only be directional if you have two separate SfiI cut sites that match up on both the insert and the destination plasmid. The enzyme doesn't *cut* asymmetrically (both overhangs are the same length) but the sequence it cuts at isn't part of the palindromic recognition sequence. The arrows in the image (from NEB) I added indicate the cut sites on either strand. N is any nucleotide. There are also enzymes like BsaI used for Golden Gate cloning that recognize a palindromic site but cut off to the side of it by a few basepairs in a similar concept, except Golden Gate usually takes advantage of the fact that you can get the enzyme to remove its own recognition site entirely and leave a "scarless" ligation in many cases.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning