User:Anthony Salvagno/Notebook/Research/2009/10/26/SDM Preplanning

I am basically starting from scratch starting tomorrow. I need to get the unzipping construct working. So for the immediate future I will be doing that. Eventually I will need to go back to Osley lab and get the Shotgun clones and ligate them in. But before that I will use pBR322 as my sample DNA. Below I will plan out my steps for achieving all this. Right now the anchor creation is the most immediate reaction so that will have the most detail, but I will do a miniplan before I do anything else throughout the week, typically the day before. Please leave comments for feedback, because that is the point of this preplanning page.

Anchor

To be performed: 10/27/09
This is created from PCR of a plasmid, is about 1.1kb, and needs to be digested with BstXI to produce an overhang complimentary to the Adapter.

Supplies

1. Primer - 10uM pRL574-R1985 (Steve Koch 22:12, 26 October 2009 (EDT): I think it's worth making new 10 uM dilutions from the 100 micromolar stocks)
2. Primer - 10uM pRL574-F834D
3. 1:100 pRL574 - old stuff (Steve Koch 22:12, 26 October 2009 (EDT): Slight chance that it's too old, but I don't think so, since it worked in some lanes)
4. NEB 10x Standard Taq Buffer (4 tubes)
5. NEB Taq (2 tubes)
6. 50mM MgCl2
7. 10x PCR Mix -MgCl2 Invitrogen (Steve Koch 22:12, 26 October 2009 (EDT): I am pretty sure that I always used minus MgCl2 in the past. The reactions are super-sensitive to MgCl2 concentration, so this is something to keep your eye on. As in 2.5 mM behaves differently than 3 mM. So, make sure that you're using exactly the right concentration, and that your buffers are very well mixed after thawing.)
8. 2.5 mM dNTP Mix (2 tubes) (Steve Koch 22:12, 26 October 2009 (EDT): I am worried about using this (just because it's a new parameter). To be more comfortable, make sure that by only diluting it 10x, there won't be weird things left over (such as EDTA) from the storage buffer. That is: see what the storage buffer is for 25 mM dNTP mix (which results from 100 mM individual) and compare with the 2.5 mM mix)

I have lots of stuff of everything so quantity is no problem. The Standard Taq buffer from NEB comes with MgCl2 in solution, so I don't need to add more. If initial reactions don't work I suppose I can use my 50mM stock to adjust the MgCl2 content. Typically I have about 250uM dNTPs in solution starting from 25mM, but now I'm starting from 2.5mM so it is basically 10x.

Recipe

The following recipe is based on standard protocols from NEB and Invitrogen. I will work off of this unless someone says otherwise.

^{SJK 22:23, 26 October 2009 (EDT)}

22:23, 26 October 2009 (EDT)
TASK: Use the thermocouple (and possibly DAQ card) to verify that the thermal cycler is working. This is akin to what Linh was doing to make sure the +3C e-ice buckets were working. I'd guess that Andy knows where it is. Perhaps Brian would like to help with this?

Also, make sure to link to your thermal cycler program (name and spreadsheet showing what it's programmed to do)

{{#widget:Google Spreadsheet |key=tkyVmiQqmKjoQ9PLJOSRJmw |width=800 |height=300 }} {{#widget:Google Spreadsheet |key=tQItPDIPjA7sJm0q4VWqLAQ |width=500 |height=300 }} (Steve Koch 22:16, 26 October 2009 (EDT): What about the mixing process? Since all tubes are the same, will mix the master mix in a 1.5 mL tube, including all components, with Taq added last. Keeping everything cold until putting tubes in the machine with hot lid pre-heated... other details?

Also, looks like you have too little dNTPs? 8 ul instead of 10 ul?)

Digestion

After PCR I will need to digest the products and then gel extract the result. The cut site for BstXI is like 20bp from the 3' end of the fragment so in order to visualize this I will have to do a PAGE to make sure the reaction is working. I suppose I could run a regular gel and if I do it for long enough, 20bp should be visible. This would be better because I don't remember how to do PAGE and I don't want to do it now. (Steve Koch 22:18, 26 October 2009 (EDT): The regular gel won't get you a definitive answer. If you're worried, you can use another plasmid (with a BstXI site) digested in another tube, to at least confirm that the BstXI is active. Otherwise, I think the diagnosis by ligation idea we came up with is the best--easier than PAGE I think)

Adapter

To be performed: 10/28/09

^{SJK 22:21, 26 October 2009 (EDT)}

22:21, 26 October 2009 (EDT)
Yes, that's the right buffer. The salt is to help hybridization. I highly recommend starting over with this step, and especially re-measuring the concentration of the stock oligos (since you suspected the nanodrop didn't work over at Osleys). Almost always (maybe always) the certificate of analysis is very accurate and the concentration of suspended oligos is within 25% of that expected, I'd say. Also, you should use the thermal cycler program to slowly cool the oligos.

As far as I know there isn't a crucial way to anneal these things. You need equimolar amounts of each oligo (Top and Bottom are the names) and some annealing buffer. I made some annealing buffer some time ago but didn't write down my recipe. I'm pretty sure I got it from here: Koch's Recipe
Other than this I don't really know what I should do. I also only have the formerly annealed adapter here so I would have to melt and reanneal, but if I didn't add components properly then I would be SOL.

I can use PAGE here to determine how the reaction is working because I would either have short dsDNA or lots of ssDNA. By this point I would have to take the time to learn how to do this setup.

Plasmid Digestion

To be performed: 10/29/09
This is a rather easy protocol. It all depends of reaction size and plasmid amount. Basically I want to digest a ton of plasmid with EarI, run the result through a gel, extract the large band, and dissolve the gel. Then I'm ready for ligation.

Right now I think I have about 20ug of pBR322 left. I'm wondering if I should just digest the rest of it or hold off. If we aren't going to do transformations for a while, then I would want to save a little bit for that at the very least. This would kill all my supply and I would have to use in moderation or buy more. (Steve Koch 22:24, 26 October 2009 (EDT):You should buy another $50 tube now so it's on hand.)

Ligation

To be performed: 10/30/09
If I get to this this week (which should be possible) I would just place each product (anchor, adapter, plasmid) in solution with T4 Ligase and 10x buffer and let it go to work. I will do a specific recipe preplanning when I get to this stage.