User:Anthony Salvagno/Notebook/Research/2011/02/17/pALS dual construct thoughts and more

From OpenWetWare

Jump to: navigation, search

Quick Navigation: Pages by Category | List of All Pages | Protocols

I'm calling it the dual construct because it can serve as both unzipping anchor and as stretching DNA right out of the PCR. I'm also calling this page what it is because I don't really know how to organize it and I don't care. Damn my future self when needing precious information that could be contained in these pages.

Contents

Yesterday's PCR

Looks like I took a step back a little bit. The PCR didn't work as expected with the perfect match and there was as much 3kb band as there was previously, but the smeariness did go away. I think I will raise the reaction temp a couple of degrees and hope that gets the job done. In the meantime I will purify this reaction and digest it as below.

pALS BstXI Digestion

View/Edit Spreadsheet

Nanodrop:

  • 170ng/ul --> 59nM

PCR 2

View/Edit Spreadsheet

Same reaction as yesterday.

Possible Experiments

Walking down the hallway I thought to myself that overstretching, while being a useful experience is rather boring. Then I wondered how I could make it better and more exciting and possibly useful. Then it dawned on me that we have enzymes and DNA that those enzymes can stick to. Then it dawned on me that we possibly have everything we need to possibly do a cool experiment. And do we shall (one day soon). I need to think on this for a while and confer with Koch more, but the potential is here.

Talking with Koch here are things we discussed:

  • getting enzyme to bind to ends would prevent initial denaturing and could reveal a cool pop-off effect. One that no one may have ever seen/done.
  • could try something simple like BstXI binding to one end and see what happens
  • also could try not pulling all the way, returning trap to start and pulling again to see if we can replicate the force curve.
  • Could try overstretching and doing above experiment with sybr safe stain since it intercollates into the DNA.

Koch and I talked about rate constants and trying to figure out how much enzyme to load into solution. Since companies publish the stock as units, there is no way of knowing really what concentration is in 1ul so I will have to just guess and play around, unless I can find out somewhere.

Personal tools