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=X3 Dilution Test= Based on the results from the X2 dilution test, I have a couple of factors I'd like to change for testing X3. From the gel results yesterday, I'd say that I couldn't see signal below 10^9 molecules, which isn't great. However, I'm guessing that this is probably due to loss of bead signal (I lost too many beads). So, I'm going to do 2 things: (1) double the number of beads. This won't actually do anything except make it easier for me to see the beads. The number of beads shouldn't change the results, only the number of targets. (2) Technical replicates. Sometimes n=1 isn't a great idea. So I'll do n=3. And finally, (3rd bonus point!) I'm going to do fewer samples and only test in the higher numbers (0-5 which ranges from 1E11 to 1E6 molecules, respectively). ==Buffers== '''KLN Mix''' {| 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="180" height="31" | Reagent | width="65" | Stock | width="65" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase Buffer | align="center" valign="bottom" | 10X | align="center" align="center" valign="bottom" | 3 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | dNTPs | align="center" valign="bottom" | 100 uM | align="center" align="center" valign="bottom" | 3 |- style="font-size:12pt" | height="15" valign="bottom" | Hemo Klentaq | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 6 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" valign="bottom" | 5 U/uL | align="center" align="center" valign="bottom" | 3 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 15 |} ==Protocol== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | width="85" height="27" | Tube No (A-C) | width="83" | Dilution | width="68" | Molecules Available |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 100000000000 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10000000000 |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 100 | align="center" align="center" valign="bottom" | 1.E+09 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 1000 | align="center" align="center" valign="bottom" | 1.E+08 |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 10000 | align="center" align="center" valign="bottom" | 1.E+07 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 100000 | align="center" align="center" valign="bottom" | 1.E+06 |} <ol> <li>Latch and Padlock Hybridization</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 oligo per sample to bead solution; incubate at RT for 5 min</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube</li> <li>Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes</li> <li>Chill on ice for 3 minutes</li> <li>Add sample to prepared strep beads; vortex to suspend</li> <li>Incubate for 30 min at 37C with intermittent mixing</li> <li>Apply magnet and remove supernatant</li> <li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li> <li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol> <li>Circularization</li> <ol type="A"> <li>Prepare 30 uL KLN mix</li> <li>Add 2 uL KLN mix to 20 uL of reaction</li> <li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8-30|tomorrow]] </li> </ol></ol> [[Category:ComboLock]] [[Category:20160826]] [[Category:20160829]]
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