User:Anthony Salvagno/Notebook/Research/2010/12/14/Tethering On Demand: Difference between revisions
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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. | 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. | ||
==Actual Experiments== | |||
[[Category:Ant Tethering]] | [[Category:Ant Tethering]] |
Revision as of 12:33, 14 December 2010
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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.
Other
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.