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=Double Stranded Probe Production= [[Daniel:Notebook/PosSequencing|Back to Calendar]] I need to make more of these. ==Protocol== <ol> <li>qPCR</li> <ol type="A"> <li>Make three 8.2X (1X) master mix with the following reagents</li> <ol type="a"> <li>8.2 (1) uL 10 uM AP1v4U</li> <li>8.2 (1) uL 10 uM BiotinU</li> <li>172.2 (21) uL nfH<sub>2</sub>O</li> <li>205 (25) uL 2X SYBR Fast</li> <li>16.4 (2) appropriate barcode oligo (100 pM)</ol> <li>Add 50 uL master mix to 8 lanes per sample</li> [[Image:PlateLayout-20170519-BarcodeOligoProduction.png|400px]] <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>Qiaquick Column</li> '''Note''': I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 6 columns total) <ol type="A"> <li>Pool 2 samples and 500 uL PB (5X binding buffer)</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through <li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li> <li>Let stand with cover open in fume hood for ~5 minutes</li> <li>Transfer column to a new 1.5 mL eppendorf tube</li> <li>Add 35 uL nfH<sub>2</sub>O to column</li> <li>Let stand 1 minute</li> <li>Spin for 1 minute at 14000 rpm</li> <li>Pool samples when finished; concentrate if desired</li> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 48 uL TBE and 12 uL 6X dye</li> <li>Aliquot 10 uL mix to parafilm</li> <li>Add 2 uL sample or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to appropriate lane</li> <li>Run gel at 240V for 24 minutes</li> <li>Open gel and stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol> <li>Qubit</li> <ol type="A"> <li>Mix 1194 uL dsDNA buffer and 6 uL dye</li> <li>Aliquot 190 uL to standard tubes and 199 uL to dye tubes</li> <li>Add 10 uL standard or 1 uL dye</li> <li>Vortex and spin down</li> <li>Let sit 5 minutes in the dark</li> <li>Measure in the Qubit</li> </ol></ol> ===Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FF66;font-size:12pt;font-weight:bold" align="center" | width="180" height="45" | Sample | width="100" | Qubit Conc (ug/mL) | width="100" | Amount (nM) | width="100" | Total Amount (fmol) | width="100" | Amplification (Over 40 amol) |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v1 | align="center" align="center" valign="bottom" | 20.1 | align="center" align="center" valign="bottom" | 324 | align="center" align="center" valign="bottom" | 2.6E+04 | align="center" align="center" valign="bottom" | 6.5E+05 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v2 | align="center" align="center" valign="bottom" | 16.3 | align="center" align="center" valign="bottom" | 263 | align="center" align="center" valign="bottom" | 2.1E+04 | align="center" align="center" valign="bottom" | 5.3E+05 |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v3 | align="center" align="center" valign="bottom" | 18 | align="center" align="center" valign="bottom" | 290 | align="center" align="center" valign="bottom" | 2.3E+04 | align="center" align="center" valign="bottom" | 5.8E+05 |} <gallery perrow=2 heights=300px widths=300px> File:20170519-qPCR-BarcodeOligoProduction.png|qPCR curves File:|Gel image </gallery> =Exonuclease Digestion Test= I'm going to use lambda exonuclease to digest the second strand (it can't digest the biotin strand since it has a 5' biotin) <ol> <li>Phosphorylation</li> <ol type="A"> <li>Set up 2 reactions 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" | dsDNA (0.3 pmol/uL) | align="center" valign="bottom" | (< 300 pmol) | align="center" align="center" valign="bottom" | 10 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | ATP (10 mM) | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;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="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 29 |- style="background-color:#BFBFBF;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>Prepare 2 reactions 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" | Phosphorylated Mix | align="center" valign="bottom" | (<2 ug) | align="center" align="center" valign="bottom" | 20 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | Lamda Exonuclease (10 U/uL) | align="center" valign="bottom" | 2 U/uL | align="center" align="center" valign="bottom" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 23 |- 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 80C for 10 minutes</li> <li>Hold at 12C</li> </ol></ol> Continued [[Daniel:Notebook/PosSequencing/2017-5-20|tomorrow]] [[Category:PosSeq]] [[Category:20170519]] [[Category:OligoPrep]]
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