User:Anthony Salvagno/Notebook/Research/2009/12/21/Tethering Marathon
Koch says I need to perfect the technique and so I will do just that. Now I already am pretty good at the general technique, meaning give me things to flow and I will flow like nobody's business. I just need to consistently get tethered beads in almost every field of view in a slide. I'm pretty sure the DNA is good, so my problem could lie in the beads or the anti-dig.
To make sure I will order some new anti-dig today and work off different preparations of beads. First I will make 2 new samples of beads. One sample I will try and sonicate the beads really well. In another sample I will try to vortex the beads really well. I will then tether several times with each bead prep sample and check my results taking images from a few FOV's.
I will alter the protocol as documented in the Bead Washing PDF I found from Bangs Labs on Fri. The normal protocol is 3 washes with spin-downs at 6600g for 5 min, but according to Bangs's Technote it should be 2200g for 15 min so I will try that. See here.
Turns out that washing and resuspending creates lots of clumps (at least with really old beads). Check out this gallery:
This image was taken after sonication.
This image was taken after vortexing (instead of sonicating). Seems as clumpy as sonicating.
This is a view of one surface of the sample in vortexing, but the same can be seen after sonication.
Instead of washing and sonicating/vortexing I just diluted the stock beads in water. Not dilute enough, but no clumps.
The best thing to do is just use unwashed beads diluted in water! What a surprise! Tethering try to come tomorrow. If you needed to perform a wash, you would have to sonicate a lot or vortex for a while.