Field of Science

DNA is biotin-tagged and binds to beads

The other day I cut H. influenzae chromosomal DNA with XhoI and separately with EcoRI. I inactivated the enzymes with heat (65°C for 20 minutes) and then incubated the DNAs with Klenow polymerase, dA, dG and dC, and biotin-tagged dU. Both of the restriction enzymes generate sticky ends with 5' overhangs that are 4 nucleotides long and include a dA, so the Klenow should have filled these ends in, incorporation the biotinylated dU to pair with the dA.

The only easy way I could think of to test whether the Klenow reaction had worked was to incubate the DNA with streptavidin-coated styrene beads (the same ones I'll use with the laser tweezers) and then run the mixture in an agarose gel. If the DNA has biotin ends, it will stick to the beads, which I think are too big to enter the gel. (I know the size of the beads (1.2 µ or 2.1 µ, depending on which vial I use); I'm just not certain of the exclusion limit of an 0.8% agarose gel.)
Anyway, the photo above shows that this worked. The top (1) and bottom (6) lanes are size standards. Lanes 3 and 5 are beads (1.2 µ ad 2.1 µ respectively) mixed with EcoRI-cut DNA that wasn't biotinylated. Lanes 2 and 4 are the same beads mixed with the biotinylated DNAs. You can see that a lot of the biotinylated DNA stayed in the well, just as it should. I don't know why the rest of the biotinylated DNA is smeared.

So tomorrow I'll get back to making the cells competent and persuading them to stick to a coverslip.

Note added later: I just did the math, to calculate how many beads and how many DNA fragments I mixed together. There would have been about 10^8 beads (fewer of the 2.1 µ and more of the 1.2 µ) and about 1 µg of DNA. EcoRI cuts H. influenzae DNA to an average fragment size of 6 kb, so 1 µg is about 2 x 10^11 DNA fragments. Thus the mix I loaded on the gels had about 2000 DNA fragments per bead. If about 10% of the DNA stayed in the wells with the beads, that's about 200 DNA fragments per bead, which should be plenty for the preliminary uptake experiments.

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