User:Anthony Salvagno/Notebook/Research/2009/12/22/Actual Tethering Marathon
After yesterday's failure to get unclumpy beads, I will use the one successful method and get really good nonclumps. Then I will do a bunch of tethering samples and show them all to you. Maybe make a video of a good sample and share. Wow, for a second I thought I was blogging and that people actually read this. Nope this is just my notebook and no one reads this. Sad :( (Speaking of emoticons, there is a song by Trey Songz called LOL :) and he literally sings "L-O-L smiley face").
Bead Prep
Ok so I've mixed 200ul of H2O with 50ul of beads (at whatever the stock concentration is). I'm going to sonicate and then try tethering. Let's see how this goes. I won't take a picture of the new sonicated beads just because.
Tethering
This is the first of probably a lot of tethering tries. I may only include this one template depending on several factors:
- not changing the recipe
- getting the new beads
- getting the detector (which means I'll be doing something else)
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1st Try
There were too many beads in solution. I will dilute and see what happens. There looked like there were plenty of chances for tethers and some good ones, but with the number of stuck beads it could be bead to bead tethers. Video to come later.
2nd Try
For this experiment I will decrease the amount of beads by diluting 1:5. It seemed like the amount of tethers was pretty similar to the amount in try 1. Just with less beads. Koch refers to tether:stuck bead ratio. So I would say the first two attempts had this same ratio.
3rd Try
For Try 3 I will dilute the DNA 1:10 more (previous dilution was 1:10) so final DNA concentration is 1:100 after gel extraction. I will use the same 1:25 dilution of beads from try 2. I'm writing this up kinda late, but from what I remember there were about the same number of tethers. Koch would have to comment cause I can't remember.
No DNA
I will be doing the same as try 1 just with no DNA, this way we can analyze how the beads behave in sample. With no DNA there were tons of stuck beads and a couple of unknown tethers. Some were bead-bead tethers (stuck bead is holding a free bead) and there was at least one that was unknown.
Bead Clumping
This has been the thorn in my foot. The ultimate goal is to have few stuck beads, no clumps, and visible tethers. More stuck beads could be because of too much DNA. This is because the more DNA there is the higher chance for multiple molecules to stick to one bead (this can give the appearance of stuck beads). Also buffer has a lot to do with the clumpiness. We have discovered that our clumps arise in the presence of BGB/Pop. I haven't narrowed that down yet, but will shortly. Here is what I have tried to minimize everything:
- Wash beads in Pop, resuspend in BGB/Pop, sonicate - this gives very clumpy beads, but the amount is reduced because of sonication. Without sonication there are tons of clumps. Poor amounts of tethers.
- Use stock beads, diluted in water, sonicated - this gives almost no clumps but there is a large number of stuck beads. Without sonication, there are clumps but still way better than option 1. Good amounts of tethers.
- Stock beads, diluted in water and BGB/Pop - at first there are no clumps but over time the clumpiness increases to terrible amounts.
Here is what I still want to try:
- Stock beads, diluted in 1x Pop
- Stock beads, diluted in PBS
I also will have a video of all the differing bead environments. That will come when all the testing is done.
