Transpacking all day long

I am finally going to talk about the next step in the Big Blue assay which is called the Transpack reaction. The basic idea is to "package" the DNA into to a phage (a virus that infects bacteria) to infect the E. coli and insert the DNA into the E. coli DNA; the phage will kill the E. coli within a few growth cycle. Any mutation in the rat DNA is then put into to the E. coli and so I can identify the mutant. The E. coli is grown on a huge plate and creates a lawn of bacteria on the agar. Any E. coli that has been infected with the phage with die creating a clear spot on the lawn of bacteria. Mutant are identified by a clear spot with a ring a blue (basically the E. coli can breakdown the X-gal due the mutation for a few growth cycles and then dies). The basics of the Transpack reaction is this: I add the isolated DNA to a red tube (this is how what it is called in the manual, you have to buy the tubes from Stratagene) and then incubate it for 90 minutes. Then I add some of solution for a blue tube (again the official name in the manual) and then incubate it for 90 minutes. I forgot to mention that there are two different agar that I use a top agar and a bottom agar (the composition varies slightly between the two). Before I start the Transpack reaction, I make petri dishes that have bottom agar in them. After the second incubation is over I infect diluted E. coli with the Transpack reaction and incubate the mix for 15 minutes in a 5ml tube. Once the 15 minutes are up, I add 3-4 ml of cooled top agar to the mix, pour it on top on the bottom agar in the petri dishes, invert it and incubate it over night. The next day, I count the number of plaques (clear spots) and that determines the efficiency of the transpack reaction and also the number of big plates I need to pour to identify the mutants.


"Yeah, yeah, everything is one way then it's the other." -Hank Hill