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AlanFung:LabNotes/Probe/2009-4-23
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==Step 4: Adapter Removal== Exonuclease treatment '''Done on 4/22/09''' *'''Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube''' *'''Add in (1/10 of total volume) 6uL 10X Exo buffer''' *'''Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR''' Purify the reaction with Quaquick columns * Add 5X sample volume of PB buffer into each tube, mix well * Load mixture onto column * Centrifuge at 14,000rpm for 1min * Discard flow through and add 750uL PE buffer to each column * Repeat Centrifuge * Discard flow through * Repeat centrifuge * Transfer columns to clean 1.5mL tubes * Place the column/tube assemblies on bench top wait for 2min * Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min * Repeat centrifuge * Measure concentration with Nanodrop *'''Tube A (04/22/09-1) 260/280 1.9 81.4ng/uL''' *'''Tube B (04/22/09-1) 260/280 1.81 74.4ng/uL''' *'''Tube C (04/22/09-2) 260/280 1.85 73.1ng/uL''' *'''Tube D (04/22/09-2) 260/280 1.88 69.63ng/uL''' *'''Tube A (04/22/09-1) 260/280 1.84 7.9ng/uL''' *'''Tube B (04/22/09-1) 260/280 1.78 8.5ng/uL''' *'''Tube C (04/22/09-2) 260/280 1.66 8ng/uL''' *'''Tube D (04/22/09-2) 260/280 1.76 6.4ng/uL''' * Concentration too low, probably due to over digest by Lambda Exo , next time run 24 wells to one tube *Combine Tube A(4/21) B(4/21) D(4/22-2) '''[[TUBE A]]''' *Combine A(4/22-1) B((4/22-1) C(4/22-2) by qiaquick column '''[[TUBE B]]''' * pool 3 tubes into one *add in 750uL PB buffer mix by repeat pipetting *load 450 mix to column *Spin down @ 14,000 rpm 1m *Discard flow through *Load the remaining 450uL into column *spin down @14,000 rpm 1m *Add in 750ul PE wash buffer *Spin down @ 14,000rpm 1m *Discard flow through *Repeat centrifuge *Place into new collection tube *Leave air dry for 5 m *add in 50uL DDH2O *Sit for 5m * spin down at 14,000rpm for 1m *Measure with Nanodrop *Decided to restart experiment, with alice's primer and new tube of lambda exo ==Step 1: PCR reaction system setup 48 wells== *Thaw Econo Taq on ice *Prepare strips and tubes on cooling rack *Add in Content in table to tubes accordinly {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''X24''' | align="center" style="background:#f0f0f0;"|'''X48''' | align="center" style="background:#f0f0f0;"|'''X72''' | align="center" style="background:#f0f0f0;"|'''X96''' | align="center" style="background:#f0f0f0;"|'''1.5 mL Tube''' |- |Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|9.6uL||align="right"|14.4uL||align="right"|19.2uL||align="right"|2.4uL |- |Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|650uL |- |AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL |- |RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|600uL |- |50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL |- |Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|4848uL||align="right"|7272uL||align="right"|9696uL||align="right"|1212uL |} *Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq *Aliquot 105uL using P200 pipette to strip tubes on cooling rack, leave unused tubes on cooling rack *Seal strips with strip caps *Place into Real Time PCR *Perform 94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold [[Image:20090423_CPGD40809.jpg]] ==Step 2: Probe purification with ethanol precipitation== *Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Needed''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- |100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL |- |Sample||align="right"|Sample Volume||align="right"|2400uL |- |Total Volume |colspan="2" align="right"|8648uL |- |} *Mix all content by repeat pipetting using multichannel pipette *Pool all 24 wells PCR product into tray using multichannel pipette(P300) *Mix all content by repeat pipetting using multichannel pipette *Aliquot about 3750uL to 2 x 15mL tube *Store in -80C for more than 20 mins or forever *Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge *Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C *Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' *Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube *Centrifuge at 10,000rpm for 5min at 4C *Remove all liquid, air dry sample for 5min or until samples dried out *Resuspend the DNA with 100uL RNAse free H2O *Break pellet by repeat pipetting *store in 4C overnight ==Step 3: Purify the reaction with Qiaquick columns== *Add 5X sample volume of PB buffer into each tube (500uL) *Mix by repeat pipetting *Load mixture onto column *Centrifuge at 14,000rpm for 1min *Discard flow through and add 750uL PE buffer to each column *Repeat Centrifuge *Discard flow through *Repeat centrifuge *Transfer columns to clean 1.5mL tubes *Place the column/tube assemblies on bench top wait for 2min *Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min *Repeat centrifuge *Measure concentration with Nanodrop
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