Daniel:Notebook/ComboLock/2017-2-6: Difference between revisions

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>Djacobse
(Created page with "=Version 2 Oligos Round 2 (Started Thurs Feb 2nd)= Back to Calendar ==Results== Looking back at the res...")
 
>Djacobse
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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.
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>
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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.

Revision as of 22:01, 6 February 2017

Version 2 Oligos Round 2 (Started Thurs Feb 2nd)

Back to Calendar

Results

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.

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.