I'm (finally) starting an experiment. This is the test of whether cells can take up intact closed circular plasmid DNA. The original experiment (Kahn et al 1983 - pdf here - compare lanes A and G of Fig. 3) used plasmids that had been cut, labeled with 32-P, and religated; the religation trapped some supercoils, allowing the gels to show that supercoiling was preserved in the DNA that had been taken up. Our new 32-P won't arrive until sometime next week, so I'm going to do a practice run using cold DNA. If I'm lucky, the cells will take up enough that I can see it in a gel.
The post-doc showed me the stock of the plasmid I'll use (pUSS-1, left by a previous post-doc). So this afternoon I'm just running a quick gel to check the state of this DNA. And pouring some plates to streak out the wildtype and rec-2 cells I'll be using. If the DNA is supercoiled I'll try it out tomorrow on some wildtype competent cells I have frozen.
We've also ordered the tester-kit of four kinds of streptavidin-coated magnetic beads with different surface properties. They're much bigger than I wanted, but I figure I can at least use them to find out whether H. influenzae cells stick to any of (or all of?) these beads in the absence of DNA. If they do, the planned experiments may not work.
_(cropped).jpg)
3 weeks ago in Genomics, Medicine, and Pseudoscience
What kinds of surface properties?
ReplyDeleteFrom our work a few years ago there was no binding between the cells and any of the beads we had at the time (streptavidin, carboxy, G-protein, ...), so I'd naively guess you'll find the same thing (which now will be the _good_ result!).
That's good to know (be reminded off). But these are magnetic beads, and I have no idea what their surface properties might be (except for the streptavidin). I don't suppose you know of any sources of tinier beads with streptavidin or another coupling molecule...
ReplyDelete