Daniel:Notebook/ComboLock/2017-2-6
Version 2 Oligos Round 2 (Started Thurs Feb 2nd)[edit]
Results[edit]
Looking back at the results from Friday, it would appear that none of the lanes show anything, not even the sample. So I did some digging, and I found this paper, which describes a more stable variant of streptavidin, including tests on the thermostability of biotin-conjugated oligonucleotides and streptavidin (as well as their new traptividin variant). The most relevant figure is examined in the gallery below.
- Chivers-Fig2A-StrepWestBlot.png
Western blot showing the streptavidin monomer and tetramer at various temperatures (3 min). Note that the tetramer starts degrading around 65-70C and only gets worse.
- Chivers-Fig2A-MonomerPct.png
Bar graph showing the calculated percentage fraction for the blot shown to the left. (1) and (2) indicate technical replicates
- Chivers-Fig2B-BiotinBinding.png
Western showing biotin-conjugated oligonucleotide binding at different temperatures (3 min). Percentage of free biotinylated DNA is given underneath each lane. Note that at 70C all biotin is free.
My conclusion from this is that I should rewrite the protocol to reduce the temperature of the denaturation steps. For example, I think the 95C incubation prior to latch/padlock hybridization (step 5) almost certainly denatured the streptavidin permanently. With the following wash steps (where I removed more of the supernatant than usual), the biotinylated template and all molecules bound to it likely floated away. Note that this is really only a problem in the positive control which relies on biotin/streptavidin beads. In the cells (especially those fixed in a polyacrylamide matrix) will be more thermostable.