I did a test of bead clumping. I started with my prep of chromosomal DNA that had been cut with XhoI and biotin-tagged at both ends of the fragments. I then cut this DNA with EcoRI, which will convert many of the fragments into shorter fragments with biotin-tags at only one end. Then I incubated this DNA with beads overnight, using either a very concentrated mixture (20 µl each of beads and DNA, in a total volume of 100 µl) or a 50-fold more dilute mixture (40 µl each of beads and DNA, in a total volume of 10 ml). The next morning I found almost no clumping, even less than at the beginning of the incubation.
But I had made one other change, in addition to the EcoRI digestion, which caused them to be much more vigorously mixed. Previously I had been placing the tubes containing the mixtures into the slots on our roller wheel, where they would rotate around their long axes with their bottoms always slightly lower than their tops. This allowed some of the beads to eventually settle at the bottom of the tube. This time I taped the tubes onto the sides of the wheel so they would rotate around their short axes, being turned completely upside down and then right side up with every rotation of the wheel. I don't know if this improved mixing also contributed to the elimination of clumps.
I had done these incubations with both magnetic Dynabeads and polystyrene beads. About half the Dynabeads went missing somewhere along the line (or maybe I accidentally put in less than I had intended). I tested using a hemocytometer to directly count the number of beads in a defined volume. This worked well, even though it's designed for cells much bigger than my beads, and I now know that I have about 2 x 10^7 of each type of bead, coated with DNA. But I don't yet know how much DNA is on the beads, and I'll have to use up a substantial fraction of the beads for the Picogreen assay to measure this.
I also grew up the strain carrying the DNA construct plasmid. It grew fine on a LB+Amp (ampicillin) plate, but not in LB +Amp broth. But I now suspect I may have put too much Amp into the broth.
Welcome to the 4th Reich part 1.
4 hours ago in Angry by Choice