Tuesday, January 02, 2007


So, for the last couple of months I have been trying to insert a gene into a DNA vector that contains another gene we are using. The gene already in the vector likes to rearrange it's sequence because it the same sequence repeated twice. Cells don't like repeated genes and tend to try to get rid of them. I have been using E. coli specially designed to resist rearrangements. To insert genes into the plasmids/vectors we use restriction enzymes to cut the vector at a specific site where we can then insert the gene that has ends compatible with the cut site. The thing is, putting these vectors together is like a puzzle. If you cut your vector too many times you won't have the pieces you need. If you don't have a site in the DNA that you need to have to do a lot of manipulations to get everything arranged in the correct order. I am not sure if this is making sense to anyone else at all.

Ok, back to my vector. I have been trying insert a gene into a vector using two restriction enzymes. Several of my clones had the same exact problem with their sequences and I thought that was really odd since recombination is generally a little more random. I was given a map of the vector I am trying to insert this gene in when I started so I could tell which restrictions enzymes to use. My map was incorrect. There was a site for one of my enzymes in the gene that is already in the vector. That site was not on the map. this whole time I have been thinking the vector was rearranging itself therefor making it incorrect, when really I was cutting it in more places than I wanted to be. At least I know I haven't made a mistake with what I have been doing. I probably should have caught the mistake, but I trusted the map I was given. I have the sequence for everything so I should be able to make it work, but I am still frustrated. I am going to draw a new map with all the pertinent information so I don't have this happen again when I try to put another gene in later.

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