User talk:Anthony Salvagno/Notebook/Research/2009/05/04/pBS with no insert Digestion

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Steve Koch 16:28, 4 May 2009 (EDT): Cool. This will come in handy for "calibration" of the SDM. We'll probably want to ligate a hairpin on the downstream end at some point (after ligating to the anchor). This will then allow us to calibrate the FJC model for single-stranded DNA of known length and extract FJC parameters in the range of > about 17 pN to overstretch transition (65 pN-ish). So, a good "to do" list item is to find another non-palindromic cutter which is pretty close to SAP (or rather, far away from SAP, so far around the circle that it's close). Having hard time describing that in words. Basically, with a single SapI digest, you have a linearized plasmid

  • Now I realize that we don't need to double-digest. A lot of your SapI plasmid will re-circularize, but we don't care, since you can overload the reaction with it (right?). After the ligation, you'll have a different SapI sticky end downstream that we can ligate a hairpin to. So, we'll want to at some point purchase that hairpin. I'll show you how to design it.