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=Positive Control Amplicon Production= ==Protocol== <ol> <li>Phosphorylation</li> <ol type="A"> <li>In a 0.2 mL tube, add ingredients according to table</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="153" height="36" | Reagent | width="73" | Stock Conc | width="90" | Final Conc./Amount | width="65" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | AmpLigase Reaction 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" | PCAmp2 | align="center" | 100 uM | align="center" | 1 nmol 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> </ol> <li>Ligation</li> <ol type="A"> <li>Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)</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="162" height="45" | Reagent | width="71" | Stock Conc | width="79" | Final Conc./Amount | width="65" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | AmpLigase Reaction Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 4 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 100 uM | align="center" | 500 umol | align="center" align="center" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | 5' Amplicon Oligo | align="center" | 100 uM | align="center" | 500 umol | align="center" align="center" | 5 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 5 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | Phosphate Reaction | align="center" | NA | align="center" | NA | align="center" align="center" | 10 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 25 |- 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" | 50 |} <li>Add amplicons according to following table</li> {| 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="65" height="45" | Sample | width="65" | PCAmp1 | width="65" | PCAmp3 | width="65" | LatchX2 | width="65" | LatchX3 |- style="font-size:12pt" | height="15" align="center" | AmpliconX1 | align="center" | X | align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" valign="bottom" | |- style="background-color:#D9D9D9;font-size:12pt" align="center" | height="15" | AmpliconX3 | align="center" | | X | align="center" | | X |} <li>Heat reaction to 95C for 5 min</li> <li>Lower temp to 55C</li> <li>Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix</li> <li>Incubate at 55C for 2 hours</li> <li>Heat to 95 C to denature dsDNA <li>Purify with ssDNA column</li> </ol> <li>ssDNA Column</li> <ol type="A"> <li>Add 100 uL '''Binding Buffer''' to the sample; mix well</li> <li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li> <li>Add 150 uL 100% EtOH to flow through; mix well</li> <li>Transfer to '''IC Column''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 400 uL '''Prep Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 700 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 400 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Centrifuge empty column at 14000 rpm for 2 minutes</li> <li>Transfer to empty 1.5mL centrifuge tube (low bind)</li> <li>Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute</li> </ol> ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | width="103" height="27" | Sample | width="77" | ng/uL ssDNA |- style="font-size:12pt" | height="15" valign="bottom" | C1-C2 Amplicon | align="center" align="center" valign="bottom" | 287.6 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | C3-C2 Amplicon | align="center" align="center" valign="bottom" | 331.6 |} ==Protocol (Part 2)== <ol start="4"> <li>TBE Gel</li> <ol type="A"> <li>Mix 32 uL TBE and 8 uL 6x Gel Loading dye</li> <li>Aliquot 10 uL for each sample to parafilm</li> <li>Add 2 uL of diluted sample/ladder to the drops</li> <li>Mix with the loading pipette and add 10 uL to each lane</li> <li>Run for 25 min at 250V</li> <li>Stain with 3 uL SYBR Gold for 3 min</li> <li>Rinse and image (see gallery below)</li> </ol></ol> [[Image:2016-08-23-PosConAmplicon.png|500px]] <ol start="5"> <li>Size Select</li> <ol type="A"> <li>For sample lanes, mix 16 uL sample, 14 uL 6X loading dye and 50 uL TBE (80 uL total)</li> <li>For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)</li> <li>Add 20 uL to each lane (2 gels total, 1 per sample)</li> <li>Run gel for 25 minutes at 250V</li> <li>While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube</li> <li>Stain gel for 3 minutes with 3 uL SYBR gold</li> <li>Image in gel doc</li> <li>Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)</li> <li>Image in gel doc post-extraction</li> <li>Centrifuge tubes at 12000rpm for 1.5 minutes</li> <li>Discard 0.5 mL tube and resuspend in 500 uL TE buffer</li> <li>Incubate at 37C for 1 hour with vigorous shaker</li> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> <li>Extract the supernatant and place into NanoSep column</li> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Move sample to a fresh 2 mL tube</li> <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue</li> <li>Store at -80C overnight, continued [[Daniel:Notebook/ComboLock/2016-8-24|tomorrow]]</li> </ol></ol> [[Category:ComboLock]] [[Category:20160822]] [[Category:20160823]]
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