So yesterday I incubated some streptavidin-coated polystyrene beads (2.1 µ diameter) with some biotin-tagged EcoRI-cut H. influenzae DNA. How many beads? About 3.5 x 10^8. How much DNA? About 2 µg. The mixture was incubated at 37 °C for about 4 hr, first undiluted and then diluted to 50 ml in TE buffer. I washed the unbound DNA off the beads by drawing the mixture through a filter with 0.2 µ pores; I expected this to retain the beads but allow the unbound DNA to pass through. I washed the filter by drawing 25 ml of TE buffer through it, five times. This was a slightly less thorough series of washes than it sounds, because I tried not to always leave a little bit of buffer on the filter, worrying that if it was sucked dry the beads might be difficult to recover. But I didn't always succeed so the washes were pretty thorough. The thoroughness of the washes becomes important below.
I put the filter into a tube with a ml of THE and agitated it a lot to resuspend as many of the beads as possible. I then used the hemocytometer to count the resuspended beads and a comparable input bead suspension. This showed that I'd recovered more than 90% of the beads. Then I used Picogreen to measure the amount of DNA in the bead suspension: 330 ng/ml. This let me calculate how much DNA was on each bead: about 1000 kb! The average fragment size of EcoRI-cut H. influenzae DNA is about 3-4 kb, so this is about 300 DNA fragments per bead.
I was initially quite pleased with this result, but then started worrying that this was to high to be true. Is there even room on a 2.1 µ bead for this many fragments? And 1000 kb is more than half a H. influenzae genome. I rechecked my calculations, and they all seemed correct. I had never tested whether the filter-washing procedure worked as I thought it should - might much of the DNA have been trapped on the filter rather than being wwashed away, and might it then have been resuspended along with the beads? If so, most of the DNA in my washed beads prep might not be bound to the beads
So this morning I pelletted the beads, removing all but ~20 µl of the supernatant before resuspending them in another ml of TE, and did Picogreen assays on both the bead-free supernatant and the resuspended beads. This showed that about 75% of the DNA was indeed on the beads. This means I have lots of beads with lots of DNA on them, ready for many tweezers experiments.
Yesterday I also used the washed beads to transform competent cells. Preliminary colony counts (the plates need longer incubation) suggest that the transformation frequency was very low (about 10^-7), much lower than expected for the presumed DNA concentration of ~100 ng/ml. This is consistent with much of the DNA being inaccessible to the cells. (But I should go back and check a control transformation I did in another experiment, in case the EcrRI-cut DNA always transforms poorly*.)
* Indeed, EcoRI cuts within the gyrB (NovR) gene, and even when the DNA was not bound to beads the transformation frequency was only 1.3 x 10^7.
Real tough guys - lichens
3 hours ago in The Phytophactor