User:Anthony Salvagno/Notebook/Research/2009/12/29/Tethering with New Beads and New anti-dig
Last week we got both new microspheres and new anti-dig. The microspheres are none fluorescent and are .97um in diameter. The anti-dig is the same. I aliquotted the anti-dig in 20ul amounts, flash freezed them, and then stuck them in the -20C freezer. There was some unknown amount left in the bottle and I am using that today. I estimated like 8ul, so I did a 1:10 dilution to use for tethering.
I am doing 2 samples of dsDNA. One sample is using the old anti-dig, and the other is using the new.
The incubation times of the anti-dig and initial BGB are a little longer then specified by an unknown amount of time, but not more than 2 min each. This is because I was looking stuff up while tethering and decided it was ok to let it sit.
Ok so both samples failed to achieve results. It seems the big beads (not washed and diluted in H2O) don't stick and don't make tethers. There are several things I would like to try now:
- Dilute big beads in BGB/POP
- Mix big beads with little beads
I will probably end up doing like 4 samples this afternoon. I will do a sample of tethering:
- just little beads
- big beads in BGB
- big bead/little bead mixture
Ok so that's three. If none of those work I will try to tether with either Brian's samples, or the unzipping DNA. I'll see how far I get later.
I now have 4 samples of beads to choose from:
- Big beads in H2O (1:5 dilution)
- little beads in H2O (1:25 dilution)
- big beads and little beads in H2O (from dilutions above)
- big beads in BGB/POP (1:5 dilution)
I admit the image is a little sperm like but it defines what the tube is quite nicely I think. Anyways, on to the tethering...
Results Part 2
So there were no tethers of big beads in either sample, but I did get some steps forward. In the sample with BGB beads, I actually got some stuck big beads. In the sample with the mixed beads, I had tethers with the little beads. So it definitely seems like something is wrong with the big beads. Also the beads look like they are more than 2x the diameter of the small beads.