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=Bolt Test (Started [[Daniel:Notebook/ComboLock/2017-3-8|Wednesday March 8]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-No Ligase== '''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="210" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 6 (AB) | align="center" valign="bottom" | No Ligase |} <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 following into a 0.2 mL tube</li> <ol type="a"> <li>4 uL each PCCA-03 and PCCB-03 (10 uM each)</li> <li>12 uL wash buffer</li></ol> <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</li> <li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Repeat wash step above</li> </ol></ol> ==Protocol-Part 2-LB hybridization & ligation== <ol start="3"> <li>Bolt and Latch Hybridization/Ligation</li> <ol type="A"> <li>Set up the following master mix</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | width="125" height="30" | Reagent | width="65" | Stock Conc | width="65" | Final Conc./Amt | width="65" | uL added | width="105" | Master Mix (4.2X) |- style="font-size:12pt" | height="30" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" | 16.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 4.2 |- style="font-size:12pt" | height="15" valign="bottom" | Lock Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 4.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 13 | align="center" align="center" valign="bottom" | 54.6 |- style="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" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 79.8 |} <li>Add 19 uL probe mix to each sample</li> <li>Incubate at 55C for 10 minutes</li> <li>Incubate at 25C for 10 minutes</li> <li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> <li>Add 1 uL T4 Ligase to each reaction</li> <li>Incubate at RT for 15 min</li> <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> <li>Wash twice with 100 uL wash buffer</li> </ol> ****** <li>Elution</li> <ol type="A"> <li>Add 50 uL wash buffer to each sample</li> <li>Cook at 95C for 15 minutes</li></ol> ****** <li>qPCR</li> <ol type="A"> <li>Make master mixes according to following recipes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66CCFF;font-size:12pt;font-weight:bold" align="center" | width="220" height="30" | Reagent | width="85" | Single Rxn | width="85" | MMC1 (6.2X) | width="85" | MMC2 (6.2X) | width="85" | MMP (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer Pair | align="center" valign="bottom" | NA | align="center" valign="bottom" | p24/p4RC | align="center" valign="bottom" | p6/p12RC | align="center" valign="bottom" | p24/p12RC |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | uL Fwd Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 6.2 |- style="font-size:12pt" | height="15" valign="bottom" | uL Rev Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6.2 | 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" | uL nfH2O | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 130.2 | align="center" align="center" valign="bottom" | 130.2 | align="center" align="center" valign="bottom" | 130.2 |- style="font-size:12pt" | height="15" valign="bottom" | uL Kapa SYBR Fast | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 155 | align="center" align="center" valign="bottom" | 155 | align="center" align="center" valign="bottom" | 155 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 48 |style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 |} <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-03132017-BoltTest-NoLigase.png|600px]] *'''Note''': The parentheses in the descriptions indicate which sample was used *'''Note''':The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green) <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x30</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 360 uL TBE, 90 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=3 heights=300px widths=300px> File:20170313-qPCR-BoltTest-NoLigase.png|qPCR curves File:2017-03-13-BoltTest-NoLigase.png|Gel image-product targets File:2017-03-14-BoltTest-NoLigase-Purified.png|Gel image-product targets-Qiaquick purified </gallery> ===Results-CANTC and Efficiency=== Take this with the [[Daniel:Notebook/ComboLock/2017-3-10|Friday results]] [[Image:20170313-BoltTest-NoLigase-SampleValues.png|600px]] [[Image:20170313-BoltTest-NoLigase-Efficiencies.png|600px]] The above graph (and Friday's, which is just this graph without the Sample-TR2 and No Ligase samples) had potentially wrong calculations for efficiency, since they only calculated the amount in the qPCR reaction (2 uL), which is a small fraction of the amount eluted (50 uL). [[Image:20170313-BoltTest-NoLigase-Efficiencies-Corrected.png|600px]] [[Category:ComboLock]] [[Category:20170308]]
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