How to add a sequence "inframe" of my gene? Help please! - (May/09/2006 )
Hello everyone, I am chemist trying to do PCR and cloning. I am designing primers for a particular domain of a gene that I want to clone. I need to add the start codon on my primers inframe of the part I want to clone; but I don't know how to do that. I know the theory behind primer desing but I dont know to make it inframe. I dont know if I have explain myself good enough, but can anybody explain about how to do an inframe insertion??
Thank you
well since the codons for each amino acid compromise 3 nucleotides, assess the part of the domain you want to PCR and ensure that once your primers amplify this area that they remain in-frame,
thus using your first AA in the sequence as a reference move upstream 3 base pairs at a time until you identify an area to use as your primer site, conruct your primers around this side including your start codon in frame, all whould be well as long as you remain in the multibles of 3
thus using your first AA in the sequence as a reference move upstream 3 base pairs at a time until you identify an area to use as your primer site, conruct your primers around this side including your start codon in frame, all whould be well as long as you remain in the multibles of 3
Thank you very much!!
Thank you
Hi. Don't forget that if you are going to clone your PCR product into a plasmid, you will need to have a restriction enzyme site on the primer. Do you know what enzyme you need? What plasmid are you planning on using?
Welcome to the nightmare, I mean the wonderful world, of cloning! May your reactions amplify, your fragments ligate, and your constructs express (kind of the protein chemists version of "Prosper and grow"...)
Oh God!! I totally forgot to follow up this question. But, thanks for your comment. I did know the enzymes I wanted to use and the vector. What I did was to add the restriction sites to the ends of the primers, then for the fw I added the start codon after the restriction site. I got my clones, they are sequenced with 100% identity so Im happy
Hello everyone, I am chemist trying to do PCR and cloning. I am designing primers for a particular domain of a gene that I want to clone. I need to add the start codon on my primers inframe of the part I want to clone; but I don't know how to do that. I know the theory behind primer desing but I dont know to make it inframe. I dont know if I have explain myself good enough, but can anybody explain about how to do an inframe insertion??
Thank you
Hi. Don't forget that if you are going to clone your PCR product into a plasmid, you will need to have a restriction enzyme site on the primer. Do you know what enzyme you need? What plasmid are you planning on using?
Welcome to the nightmare, I mean the wonderful world, of cloning! May your reactions amplify, your fragments ligate, and your constructs express (kind of the protein chemists version of "Prosper and grow"...)