User:Anthony Salvagno/Notebook/Research/2009/11/01/Preplanning for Anchor Digest
PCR Cleanup
First I need to cleanup my PCR reactions. I have 5 tubes of DNA each with about 41.6ng/ul in 88ul. That comes out to 18ug of DNA! Sweet. I will cleanup in 2 Qiaquick columns (to maximize DNA) and elute with the normal 50ul per column. You can do 30ul, but I'm running a digestion after and I don't think concentration is all that important.
- Steve Koch 21:15, 1 November 2009 (EST): I agree here. Lots of DNA, good!
- Steve Koch 21:25, 1 November 2009 (EST): Since you're planning on using entire amount in restriction digest, without much dilution, I would elute with "T" buffer, as opposed to TE. I would also make it 0.1X "T" so it doesn't mess with the digestion.
- Anthony Salvagno 22:47, 1 November 2009 (EST):What is Qiagen's elution buffer? Also, why would I want .1x T? I've never had problems with digestion from eluting with whatever Qiagen provides.
- Steve Koch 23:15, 1 November 2009 (EST):It is buffer EB, which I think is just 10 mM Tris, pH8.0, no EDTA. But you'll want to double-check that. How do you know you've never had trouble? Didn't you have some SapI inefficient cutting? It looks like in this case that buffer EB won't mess with NEB Buffer 3. But 1/10 dilution of EB would be fine, because you can elute with pure water too (of the right pH). It's always a really good idea to conisder how much you're diluting all of the components of your reaction, especially as in this case, where you're not diluting the DNA component much at all.
Digestion
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This plan is easily changable. I first need to find out how many units per ul are in my BstXI tube and then I will vary reactions accordingly (1U/ug of digesting DNA). It also depends on if I decide to elute PCR DNA in 30 or 50ul of elution buffer. I don't know if concentration is important or not, but in the essence of needing less gel lanes I could just try and squeeze it into a smaller reaction volume. Also, I don't know if I should digest all of it or not. I think I should because I will need to digest more in the future and I might not want to go through that step now.
- Steve Koch 21:25, 1 November 2009 (EST): Make sure to specify Buffer #3.
Refs
- Steve Koch 21:19, 1 November 2009 (EST) I was thinking, "shouldn't it be at 55C?" and found that NEB now has a recombinant form, starting with lot 15, that should be at 37C. But make sure you have the new lot
- Here's the general BstXI page: http://www.neb.com/nebecomm/products/productR0113.asp
- The concentration is 10 U / microliter. So you probably are using too much enzyme. I'd use 2 microliters per reaction.
Gel Extraction
I will setup and run a gel. Depending on how much reaction I have, I will either use the smaller gel apparatus or the larger one. I can prolly get away with the smaller one cause each lane can hold up to 30ul (if I use the larger lanes), so if I tape lanes and make slut lanes then I can fit more. How effective is the gel extraction from Qiagen? Should I try and fit it all in one column and assume 50% loss?
- Steve Koch 21:27, 1 November 2009 (EST): They say 80% but 50% is a better bet. One thing to worry about is how much agarose it can handle. I can't remember the answer to that. But one column is probably a good bet.
