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=RCA + Circularization Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled. ==Protocol-Part 1== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="120" height="28" | Sample | width="190" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (ABC) | align="center" valign="bottom" | Sample |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (ABC) | align="center" valign="bottom" | No Padlock PO4 |} <ol> <li>Phosphorylation-Padlock0501</li> <ol type="A"> <li>Set up reaction according to table</li> {| 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="150" height="30" | Reagent | width="92" | Stock Conc | width="91" | Final Conc./Amount | width="65" | uL added |- style="font-size:12pt" | height="30" valign="bottom" | T4 Polynucleotide Kinase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | ATP | align="center" | 10 mM | align="center" | 1 mM | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Padlock0501 | align="center" | 10 uM | align="center" | 100 pmol total | align="center" align="center" | 10 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | T4 DNA Kinase | align="center" | 10 U/uL | align="center" | 10 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 5 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |} <li>Incubate at 37C for 30 min</li> <li>Heat kill enzyme with 15 minutes at 65C</li> </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>Dilute 5 uL C4-C2 template into 45 uL nfH2O <li>Add 2 uL diluted template 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>Padlock Hybridization</li> <ol type="A"> <li>Make following 6.1X master mixes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#DA9694;font-size:12pt;font-weight:bold" align="center" | width="150" height="39" | Reagent | width="85" | MM1 (uL) | width="85" | MM2 (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Padlock0501 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 3.1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PNK Reaction | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase Buffer | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 6.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 49.6 | align="center" align="center" valign="bottom" | 52.7 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 62 |style="font-weight:bold" align="center" align="center" valign="bottom" | 62 |} <li>Resuspend sample in 20 uL padlock buffer</li> <li>Incubate at 37C for 30 minutes</li> <li>Wash twice with wash buffer</li></ol> </ol> ==Protocol-Part 2== <ol start="4"> <li>Circularization</li> <ol type="A"> <li>Prepare 6.5X uL Phusion mix</li> {| 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="190" height="30" | Reagent | width="65" | Stock Conc | width="65" | Final Amount | width="65" | 1x Vol (uL) | width="105" | MM Vol (6.5x) (uL) |- style="font-size:12pt" | height="15" valign="bottom" | NAD+ | align="center" | 5 mM | align="center" | 40 nmol | align="center" align="center" | 8 | align="center" align="center" | 52 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" | 1 mM | align="center" | 600 pmol | align="center" align="center" | 0.6 | align="center" align="center" | 3.9 |- style="font-size:12pt" | height="15" valign="bottom" | Betaine | align="center" | 5 M | align="center" | 15 umol | align="center" align="center" | 3 | align="center" align="center" | 19.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | 10X AmpLigase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 | align="center" align="center" | 13 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 10 U | align="center" align="center" | 2 | align="center" align="center" | 13 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Phusion HF DNA Polymerase | align="center" | 2000 U/mL | align="center" | 6.4U | align="center" align="center" | 3.2 | align="center" align="center" | 20.8 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 1.2 | align="center" align="center" | 7.8 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |style="font-weight:bold" align="center" align="center" | 130 |} <li>Add 20 uL phusion mix to 20 uL reaction</li> <li>Incubate at 55C for 2 hours</li> </ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)</li> <li>Add 2 uL to each sample, mix by swirling pipette tip</li> <li>Incubate for 1 hour at 37C</li> <li>Heat kill by incubating for 5 min at 95C</li> </ol></ol> Continued [[Daniel:Notebook/ComboLock/2017-2-28|tomorrow]] [[Category:ComboLock]] [[Category:20170227]]
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