Field of Science

DNA looks ready to label

Both my gels gave OK results. But I didn't really use enough DNA in the conventional gel, and the CHEF gel ran faster (or longer) than would have been ideal. But the conclusion is that both DNA preps consist almost entirely of fragments bigger than 40kb and smaller than 150kb. This is fine for my initial experiments.

The next step is to tag the ends of the DNA molecules with biotin. At most ends the two strands are likely to be unevenly broken (i.e. either the 5' end or the 3' end overhanging), so I can use Klenow polymerase to fill in the ends that have 3' overhangs. I'll use a biotinylated deoxynucleotide in place of done of the normal nucleotides (e.g. biotin-dUTP), so those ends whose 3' overhangs include an A will have a biotin. Ends lacking an A won't be labeled, nor will blunt ends and those with 5' overhangs. But this shouldn't be a problem as the presence of fragments with one or two unlabeled ends won't interfere with attachment of the biotin fragments to the streptavidin beads. I expect 3' overhangs and 5' overhangs to be equally common, and both to bed much more common than blunt ends. H. influenzae DNA us rich in As and Ts so, if all goes well, 30-50% of the ends will be labeled.

I can't do this until we get some biotin-dUTP; I'll order it first thing Monday morning.

To check whether the labeling reaction works, I'll do a test reaction that also includes a small amount of a radioactive nucleotide; we have some in the fridge that should still be good as it is labeled with 33-P which has a 30-day half-life. I'll both check for incorporation of 33-P into the DNA directly, and run the DNA in a gel and do an autoradiogram (expose it to a radiation-detecting screen) to check that most of the label is in big fragments. This is important because, although small fragments are less conspicuous in gels (longer fragment shave more DNA in them so they fluorescence brighter). their ends label just as well as those of long fragments fragments.

On Monday I'll also order the streptavidin-coated polystyrene beads needed for the next step.

1 comment:

  1. The 33-P in the fridge is from 23 Feb 2007. I've still been using it in uptake assays (just adding more labeled DNA to compensate) but keep in mind that it isn't that hot anymore.


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