Checking clones by sequencing - (Jul/04/2007 )
I am a bit confused. I want to ask you guys if you could help me make a guideline on how to analyze a sequence to make sure a gene is cloned right. I know that a BLAST search has to be done, vector contamination has to be checked, etc. Could someone make a step by step procedure of what has to be done to check results for cloning? I really aprreciate the help.
okay, I try my hand at this
1- Find out how what your gene sequence is suppose to be. One can use BLAST if the gene sequence is known, particularly if you are cloning the gene from the BAC library used in the genome sequencing project for that particular organism. However be aware that there maybe slight problems particularly in multicellular organisms, etc mice, zebrafish, humans. There may be allele difference between strains/individuals. THis will give rise to sequence differences (which may or may not be crytic) between the sequenced strain and the strain that your have cloned your gene from. It can be hard to tell if a change is natural or induced by PCR error. Only by looking at several independent isolates of your gene/plasmid can you tell which alternative is true
2- Next sequence your gene (which has already been cloned into a plasmid). Depending on how large your gene is you may need to design several primers to get a sequence read over the entire gene. The normal lenght of a sequence read is about 700bp and maybe as short as 500bp if you haven't master the sequencing trick just right.
3- compare the sequence. I use clustalW to do the comparison for me. See if there are any differences between what you see and what you expect to see.