Daniel:Notebook/ComboLock/2016-12-9: Difference between revisions
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This protocol covers a new method using ideas taken from Weibrecht ''et al'' New Biotechnology 2012 (Landegren lab). The main idea I see is that they almost never use polymerase, but prefer to instead use ligation events as the predominant form. Based on the results from the [[Daniel:Notebook/ComboLock/2016-12-1|extended latch]] experiment, the [[Daniel:Notebook/ComboLock/2016-11-8|RCA-based cell test]], and the sequencing from the first [[Daniel:Notebook/ComboLock/2016-11-1|circularization test]], it seems the probes do not circularize correctly. Note the original orientation of the C probes with the 5' end being the genome matching region and the 3' end being the latch/padlock adapter. | This protocol covers a new method using ideas taken from Weibrecht ''et al'' New Biotechnology 2012 (Landegren lab). The main idea I see is that they almost never use polymerase, but prefer to instead use ligation events as the predominant form. Based on the results from the [[Daniel:Notebook/ComboLock/2016-12-1|extended latch]] experiment, the [[Daniel:Notebook/ComboLock/2016-11-8|RCA-based cell test]], and the sequencing from the first [[Daniel:Notebook/ComboLock/2016-11-1|circularization test]], it seems the probes do not circularize correctly. Note the original orientation of the C probes with the 5' end being the genome matching region and the 3' end being the latch/padlock adapter. | ||
[[File:C_Probes-Original.png| | [[File:C_Probes-Original.png|200x100px|C probes in the orientation of their original design. Template (mRNA/control oligo) is in black, C probes in red. Vertical line indicates 5' end, arrow indicates 3' end.]] | ||
When subjected to polymerization, if the latch is not present this yields a product that follows the backbone of the second C probe. See image below for clarification. | When subjected to polymerization, if the latch is not present this yields a product that follows the backbone of the second C probe. See image below for clarification. |
Revision as of 18:36, 9 December 2016
Lock Oligo Protocol
This protocol covers a new method using ideas taken from Weibrecht et al New Biotechnology 2012 (Landegren lab). The main idea I see is that they almost never use polymerase, but prefer to instead use ligation events as the predominant form. Based on the results from the extended latch experiment, the RCA-based cell test, and the sequencing from the first circularization test, it seems the probes do not circularize correctly. Note the original orientation of the C probes with the 5' end being the genome matching region and the 3' end being the latch/padlock adapter.
When subjected to polymerization, if the latch is not present this yields a product that follows the backbone of the second C probe. See image below for clarification.