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
Content Volume X24 X48 X72 X96 1.5 mL Tube
Jan_09_CPG_D40809 (10uM) 0.2uL 4.8uL 9.6uL 14.4uL 19.2uL 2.4uL
Econo Taq 50uL 1250uL 2400uL 3600uL 4800uL 650uL
AP1V6 AP2V6 Mix (50uM) 0.4uL 9.6uL 19.2uL 28.8uL 38.4uL 4.8uL
RNAse free H2O 50uL 1200uL 2400uL 3600uL 4800uL 600uL
50X SYBG I 0.4uL 9.6uL 19.2uL 28.8uL 38.4uL 4.8uL
Total Volume 101uL 2424uL 4848uL 7272uL 9696uL 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 File:20090423 CPGD40809.jpg