With the tweezer ready for tweezing, calibration under-way, and some last minute mechanical features being added it's time to get tethering, stretching, and unzipping going again. I need to get my head together to get it working at least as efficiently as it was before BPS last year. Yesterday I did some dabbling and it wasn't very good. Things need to be better.
- make new BGB
- I have a 50ml tube of it ready to go. It just needs to be filtered. I made this last June/July I think. Is it ok to go?SJK 21:10, 19 October 2010 (EDT)
21:10, 19 October 2010 (EDT)
I would make new stuff. I'd also consult with Andy, since he's a casein expert now, whether or not he wants to be.
- make new POP
- I definitely need new pop and I think I need to remake one chemical in the mix.
- prepare more anti-dig SJK 21:11, 19 October 2010 (EDT)
21:11, 19 October 2010 (EDT)
Do you mean dilute a new aliquot in PBS?
- Get new beads
- small beads, the big beads are fine except they don't tether well
- try cleaning with Windex
- may be something magical in the windex that gave Koch great tethering. This is literally the only thing I've yet to do from Koch's glory days.
- SJK 21:12, 19 October 2010 (EDT)
21:12, 19 October 2010 (EDT)
Well, it's embarrassing, but it may work. You could also clean by sonication or other method.
- Right now I'm using 1:100 concentration for the 1.1kb tethers I think this puts it somewhere in the 10-100pM range
- I need to mix my DNA tubes up. They've been sitting in the lab top coolers for some time now.
- For DNA I have: TpBR (tetherable pBR fragments), Tyeast (I'm calling it that for now, it's some kind of shotgun clone), 1.1kb dsDNA tethers, 4.4kb dsDNA tethers (11pM I believe)
- Since I'm starting with the dsDNA tethers (for stretching) should I use my frozen stock or will mixing it up do the trick? I guess I will try mixing first (gently of course) so I don't have to throw anything away. SJK 21:13, 19 October 2010 (EDT)
21:13, 19 October 2010 (EDT)
I think 4.4 kb is the thing to start with. But you only have 10 pM? That's not very much, especially if tethering isn't working well. It's be better to start with 10x that much if possible.