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=Version 2 Oligos Round 2 (Started [[Daniel:Notebook/ComboLock/2017-2-2|Thurs Feb 2nd]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Results== Looking back at the results from [[Daniel:Notebook/ComboLock/2017-2-3|Friday]], it would appear that none of the lanes show anything, not even the sample. So I did some digging, and I found [[Media:Chivers_Streptavidin_Variant_Stability_2010_Nat_Meth.pdf|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. <gallery perrow=3 heights=300px widths=300px> File: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. File:Chivers-Fig2A-MonomerPct.png|Bar graph showing the calculated percentage fraction for the blot shown to the left. (1) and (2) indicate technical replicates File: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. </gallery> 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 ([[Daniel:Notebook/ComboLock/2017-2-2#Protocol-Phase 2|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. [[Category:ComboLock]] [[Category:20170202]]
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