User:Anthony Salvagno/Notebook/Research/2009/11/16/Preplanning for 2 Ligations and new PCR
I have a lot of preplanning to do. I got 6 new oligos which means I have lots of ligating and some PCR to do. I need to:
- Anneal:
- Top BstXI/SapI to Bottom Biotin
- BstXI top adapter to BstXI bottom adapter
- BstXI top adapter to BstXI bottom adapter biotin
- Ligate:
- BstXI/SapI adapter to digested pBR and pRL (anchor)
- BstXI adapter to pRL
- Biotinylated BstXI adapter to pRL (to try to unzip)
- PCR
- R2342 primer with both F###-dig primers to pRL574
Annealing
I want all oligos to be equimolar in each reaction. Here is my reaction chart:
SJK 19:25, 16 November 2009 (EST)
It'd be good to put in the starting oligo concentrations and desired final concentrations in the spreadsheet to make it clear how they're equimolar. Also, would be good to write down what annealing buffer is, or to link to it. One thing comes to mind: you should make new annealing buffer. And also think about how much you're diluting these duplexes down the road. Is there too much EDTA or other items in the annealing buffer? (That is, are they getting diluted enough?)
I'm excited about seeing the upcoming ligation reactions!
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Ligating
SJK 19:29, 16 November 2009 (EST)
As far as I can see, this looks good. My only important comment is that I would add enough adapter duplex so that it is at least 2x the concentration of the outside parts (anchors) by the time you're done. Starting low (as you're doing) is good, in case you don't have as much anchor as you think. Ending high is good in case you don't have as much good adapter as you think. It'd suck to stop too low and only get a teeny amount of product that you couldn't see.
First up is the Anchor to Anchor Ligation using the BstXI double overhang adapter.
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I want to follow a similar approach to the unzipping ligation where I add adapter a little at a time until I have equimolar concentration. The difference is that this time there is no other DNA besides the anchor and adapter. Since I want to ligate anchor to anchor, my max adapter should be half the molarity that I put in of the anchor. So if I have 20nM anchor, I want 10nM adapter when all is said and done. For this particular reaction, I will add 2.5ul every 30' until the total volume is 50ul (which is 7.5ul of adapter). This gives me 15nM adapter (for 30nM anchor) at the end.
Next is the unzipping construct ligation.
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I've altered my typical reaction so that at the end of the reaction I will have 16nM adapter, meaning I will begin the ligation at 44ul total and add 2ul adapter every 30' until I am at 50ul (three more additions).
SJK 19:30, 16 November 2009 (EST)
Same comment here as above (I think you should end with more adapter)
PCR
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I will do this at the same settings I've been using with most of the same amounts of components. The only thing I am changing is the length of the product. I think my extension time is ok (2min) because you are supposed to approx. 1min for every 1kb and I am doing 1.5kb. If I am missing anything let me know. This will be done tomorrow.
I was doing some reading and now I am confused. Am I using too little template DNA? According to NEB the amount should fall in the range of .1-1ng/ul final concentration. I am starting with that amount and diluting it 100 fold. Is this too little? Conversely with Kelly, I added 15ul of 1.5ng/ul pRL574 and got successful reaction. Should I go with that amount? Some websites say you can get away with so little and others suggest more is needed. What if all my failures are a result of not having enough. I did get a couple successes, but what if I can get more? Koch?
SJK 19:33, 16 November 2009 (EST)
We don't want to start off with too much, because as you pointed out with your previous post or email, we want our final products to all have primers. If you start with 1 ng / ul, I suppose that's OK if you end up with more than 100 ng / ul of product. (Given that product is shorter than starting plasmid, that would mean that <1% of your final products would have funky ends.) I used to always use a 1:100 dilution of a plasmid midiprep. I'm looking up my notes now and if I can find the concentration of that midiprep, I'll post it here.
Steve Koch 19:43, 16 November 2009 (EST): Found my old notes. I think my 1:100 plasmid would have been 2.5 ng / ul approx. Here's the document:
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