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==Protocol== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="60" height="30" | Sample No | width="85" | Variable | width="200" | Condition |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 1 | align="center" valign="bottom" | pH | valign="bottom" | pH=9.0 |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 2 | align="center" valign="bottom" | pH | valign="bottom" | pH=10.0 |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 3 | align="center" valign="bottom" | pH | valign="bottom" | pH=11.0 |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 4 | align="center" valign="bottom" | pH | valign="bottom" | ph=12.0 |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 5 | align="center" valign="bottom" | Heat | valign="bottom" | 95.0C (3 min) |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 6 | align="center" valign="bottom" | Heat | valign="bottom" | 90.0C (3 min) |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 7 | align="center" valign="bottom" | Heat | valign="bottom" | 85.0C (3 min) |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 8 | align="center" valign="bottom" | Heat | valign="bottom" | 80.0C (3 min) |- style="font-size:12pt" | align="center" height="30" align="center" valign="bottom" | 9 | align="center" valign="bottom" | LXO (++++) | valign="bottom" | Positive Control-Lambda Exonuclease digestion |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 10 | align="center" valign="bottom" | dsDNA (----) | valign="bottom" | Negative Control-Nothing |} <ol> <li>Bead binding</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 5 uL barcode2 oligo ([[Daniel:Notebook/PosSequencing/2017-5-17|300 nM]]) and 10 uL nfH<sub>2</sub>O; incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol></ol> ===pH Denaturation Samples(1-4)=== <ol start="2"> <li>pH denaturation</li> <ol type="A"> <li>Resuspend beads in 50 uL of appropriate pH solution (see sample matrix)</li> <li>Incubate 2 minutes RT</li> <li>Magnet pulldown 1 min; remove supernatant</li> <li>Resuspend in 50 uL 1X TE buffer</li> </ol></ol> ===Heat Denaturation Samples (5-8)=== <ol start="2"> <li>Heat denaturation</li> <ol type="A"> <li>Resuspend beads in 50 uL of TE-TritonX 100 0.1% (TE-T)</li> <li>Incubate 3 minutes at the appropriate temperature according to sample matrix</li> <li>Magnet pulldown 1 min; remove supernatant</li> <li>Resuspend in 50 uL 1X TE buffer</li> </ol></ol> ===Lambda Exonuclease Sample (9)=== <ol start="2"> <li>Phosphorylation</li> <ol type="A"> <li>Resuspend beads in solution using the following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center" | width="200" height="33" | Reagent | width="100" | Final Conc. | width="100" | uL Added |- style="font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | ATP (10 mM) | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK (10 U/uL) | align="center" valign="bottom" | 10 Units | align="center" align="center" valign="bottom" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 39 |- style="font-size:12pt" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 50 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 20 minutes</li> </ol> <li>Lambda Exonuclease Digestion</li> <ol type="A"> <li>Magnet pulldown 1 minute and remove supernatant</li> <li>Resuspend in solution according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FFCC;font-size:12pt;font-weight:bold" align="center" | width="240" height="30" | Reagent | width="100" | Final Conc. | width="100" | uL Added |- style="font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="right" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Lamda Exonuclease (10 U/uL) | align="center" valign="bottom" | 2 U/uL | align="right" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="right" align="center" valign="bottom" | 43 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total | align="right" align="center" valign="bottom" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 50 |} <li>Incubate at 37C for 30 minutes</li> <li>Add 2 uL 0.5M EDTA and mix well; incubate at RT for 5 minutes</li> <li>Magnet pulldown and resuspend in 50 uL 1X TE buffer</li> </ol></ol>
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