in frame cloning - cloning (Sep/16/2005 )
pls help! how to check if my gene (insert) or in other words my cloning is in frame? i don't understand this in-frame thingy. i sent my sample for sequencing and got the sequence results, but how to check if it is in frame? hope someone can help me . thanks.
What are you trying to clone in frame? Your insert with a tag from the vector? N-terminal? C-terminal?
Did you translate the sequence results into protein?
this is a good explanation of what an ORF is, and should show you why it's important to have the correct ORF if you want to clone a gene and get a protein from it
Wow, inframe basically means that you keeping the same codon usage for translation. If you remember the addition of amino acids to form proteins from mRNA is done from 3 base recognition codons on the mRNA, if you insert/delete one base, you will affect all downstream codons. This is what you want to prevent when you add a tag to the N-term. When you initiation translation from a tag AUG start codon, the protein you a fusing needs to be within the same coding frame as the tag. Just make sure the total number of bases in the tag is a multiple of three and you wil be in good shape.