User:Anthony Salvagno/Notebook/Research/2010/12/14/Tethering On Demand
I've been trying to get tethering working for a while and so far no dice. I need to figure out what is going on here. And I may need to redo the tethering construct over from the beginning.
Experiments to Try
First I think it needs to be noted that I haven't been having good tethering efficiency. I do not think the anit-dig is the problem. I also do not think it is the beads either. That means it is somewhere on the DNA. On some days I don't get any tethers. On the days that I do, the tethers break almost instantly. What to do?
- Try different DNA batches:
- I have tons of DNA from unzipping to 1.1kb stretching DNA and 4.4kb stretching DNA
- Try different surface blockers:
- I have BGB and dephosphorylated alpha casein
That is really all I can think of doing outside of doing a new ligation reaction and PCR (which didn't work last time I did it). We shall see I suppose.
After looking at the damage done by the broken freezer, I have to reorder tons of enzymes. I should probably make new unzippable DNA anyways, this time I'll keep track of everything that I make tubes of. I still have a lot of adapters (so I won't have to do any annealing or any PCR), but I should get all the necessary pieces anyway.
Enzymes to Buy
- T4 DNA Ligase - crucial for the unzipping construct reaction.
- Taq - needed for PCR for stretching DNA
- SapI - for digestion of shotgun clones
- EarI - for digestion of pBR322
I think that is all the pressing stuff. On top of those I have to get the PCR reaction items (buffer, dNTPs, etc). Since the PCR is not going to be the first thing done, I don't have to worry right now. Once we get the freezer in I'll buy all that stuff.
In other news I think I'm going to do two Ligation reactions. This will make ligating the unzipping fragment onto the construct more efficient. More thinking will be done as time goes on.
I'm going to do experiments one at a time today and list them here as I do them:
- Unzipping DNA (from laptop cooler) tethered to small beads.
- No tethers, too many clumped beads.
- Same as (1) but beads sonicated for longer.
- Beads washed and then same protocol as (1)
- Same as (3) but in the premade perfusion chambers from Cytoskeleton