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=Mirror Probe Protocol= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==New Directions== 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. 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. <gallery perrow=2 heights=200px widths=400px caption="Original C probes, orientation and binding. C probes in red, padlock probe in green, polymerase-added bases in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end."> File:C_Probes-Original.png|Original C probe orientation File:C_Probes-BadCircularization.png|Polymerization without latch traces along the C2 probe </gallery> This actually makes quite a bit of sense since the probability of only the padlock binding is much higher than the probability of both the latch and padlock binding. ===Mirror Probes=== To address this problem, we now introduce the '''mirror''' C probes. These C probes have their 3' and 5' ends reversed such that the 3' end is the genome matching region and the 5' end is the adapter region. Notice that because of the new orientation a polymerase that has no latch to work off will fall off the second C probe, rendering it impossible to circularize (hopefully). <gallery perrow=2 heights=200px widths=400px caption="Mirror C probes have the advantage of polymerase failing to trace them by mistake. Original C probes, orientation and binding. C probe in red, mirror probe in blue, padlock probe in green, polymerase-added bases in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.> File:C_Probes-MirrorHybrid.png|Orientation of the mirror C probe (blue) File:C_Probes-Mirror-Polymerization.png|Polymerase product "falls off" the 3' end of the mirror C probe and does not create a lengthy product </gallery> To this end I have ordered two control mirror C probes to test (for the positive control oligo). ===Lock Oligos (Ligation-Based Templates)=== I have also had a second idea based off the Landegren (Weibrecht) paper. The idea is to add a ligation event that will connect together the two C probes (via the latch). This will provide a stable template for the padlock probe that becomes latch independent. First, we look at just the latch binding. Now I introduce the "Lock oligo", which is reverse complement to the latch oligo. Rather than incubating the latch and padlock at the same time, we incubate the lock and latch at the same time which ligate the C probes together. It then no longer matters if the latch remains bound to the adapter regions, the padlock has a contiguous template to work off. <gallery perrow=2 heights=300px widths=400px caption="Modified latch-padlock system integrating the lock oligo. Lock/Latch binding is ligase based-only creating a solid block to read for polymerization. C probe in red, mirror C probe in blue, latch in dashed green, polymerization-added bases in magenta, lock oligo in brown, and template in black."> File:Latch_Binding.png|Latch binding event File:LockBinding.png|Lock binding event; the lock is RC to the region between adapters (in v1 latches, the barcode+UMI) File:LockBridge.png|Lock-C probe ligation produces a "bridge" for padlock circularization File:LockPadlockCircularization.png|Padlock now binds with a complete template and can circularize/ligate for further amplification </gallery> ==Experiment== ===Sample Matrix=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="150" height="34" | Sample | width="120" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No C probe |} ===Protocol=== <ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol> <li>C Probe Hybridization</li> <ol type="A"> <li>Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li> <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add 5 uL probe mixture to beads</li> <li>Incubate at 40C for 1 hour with agitation; '''start part 3 after incubation begins'''</li> </ol> Here I realized that the Mirror probes transcript matching region is incorrect. Therefore, the experiment is canceled. ===Buffers=== '''Wash Buffer''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="65" height="30" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="65" | Amt in 20 mL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NaCl | align="center" valign="bottom" | 1.5 M | align="center" valign="bottom" | 0.5 M | align="center" align="center" valign="bottom" | 3 | align="center" valign="bottom" | 6.66 mL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | align="center" valign="bottom" | 500 mM | align="center" valign="bottom" | 20 mM | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 800 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | EDTA | align="center" valign="bottom" | 0.5M | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 500 | align="center" valign="bottom" | 40 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | nf H2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | 12.5 mL |} '''Low Salt Buffer''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="65" height="30" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="65" | Amt in 10 mL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NaCl | align="center" valign="bottom" | 1.5 M | align="center" valign="bottom" | 0.15 M | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 1 mL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | align="center" valign="bottom" | 500 mM | align="center" valign="bottom" | 20 mM | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 400 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | EDTA | align="center" valign="bottom" | 0.5M | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 500 | align="center" align="center" valign="bottom" | 20 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | nf H2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 8.58 mL |}
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