Possible to add a linker to only one blunt end? - (Jun/25/2008 )
Hi all,
I'm completely new to cloning and I'm confused by many things.
Now I'd like to clone a insert to a pIEx vector (Novagen). My insert has MscI (or NotI) and SalI sites. If I use NotI and SalI, there would be 30 extra amino acids produced besides my protein of interest. My protein is about 120 amino acids so I wonder if the 30 extra residues are too many.
If I use MscI instead of NotI, it seems there is no proper site to insert in the vector. I found linker has been used to produce sticky ends but I don't know if a BamHI linker can be applied to a insert with MscI blunt-end and SalI sticky-end.
Many thanks if you could give me some clues.
Just make one end of the linker blunt-ended and the other end a BamHI half site. that way, only the MscI site will be ligated with the linker. You may also get some conctamerisation, but you can get around that with a BamHI and SalI digest to release single-copy insert.
Just a small point about SalI, in case you haven't heard. According to the NEB website, SalI does not like cutting PCR products (but they don't go into why this should be so). You might need to purify the amplicon before you do your digestion, or you might just be lucky and it will work first time.
Thanks a lot for your reply. I don't quite understand this sentence.
For example, NEB carries BamHI Phosphorylated Linker (12-mer) (Sequence: 5' d(pCGCGGATCCGCG) 3'). Can't I directly ligate it to the blunt-end?
I read some downsides about SalI and XhoI. Will it produce similar effect on plasmid b/c I will cut out the gene of interest from a plasmid?
This cloning is really driving me crazy.
Sorry for my lack of clarity. If you are going to add something to your PCR product, it should be double stranded, right? You need to order two primers that are complementary to each other, but that also have the required restriction sites. In this case, you want one end of the adapter to have a Bam site, and the other end has to be blunt-ended. The NEB linker should work fine.
I don't think the problems of Sal I are seen in plasmid DNA.
Sorry for my lack of clarity. If you are going to add something to your PCR product, it should be double stranded, right? You need to order two primers that are complementary to each other, but that also have the required restriction sites. In this case, you want one end of the adapter to have a Bam site, and the other end has to be blunt-ended. The NEB linker should work fine.
I don't think the problems of Sal I are seen in plasmid DNA.