AlanFung:LabNotes/Probe/2009-4-23

From ZhangLabWiki
Jump to navigation Jump to search

Step 4: Adapter Removal[edit]

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[edit]

  • 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

Step 2: Probe purification with ethanol precipitation[edit]

  • Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content Volume Needed Volume
100% Ethanol 2.5X Sample Volume 6000uL
3M NaOAC 0.1X Sample Volume 240uL
Glycoblue 1/300X Sample Volume 8uL
Sample Sample Volume 2400uL
Total Volume 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[edit]

  • 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