AlanFung:LabNotes/Probe/2009-5-1

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Purpose

  • To improve my prob preping skills
  • Be extremely careful with contamination, clean every tools and equipments before use
  • Following kun's protocol exactly the way he did it on 04/16/09

Step 1: PCR reaction system setup

  • Thaw Econo Taq on ice
  • Prepare 4 strips and 2x 2ml tubes on cooling rack
  • Add in Content in table to tubes accordingly
Content Volume X32 2 mL Tube 2 mL Tube
RNAse free H2O 49uL 1568uL 784uL 784uL
50X SYBG I 0.4uL 12.8uL 6.4uL 6.4uL
AP1V6 AP2V6 Mix (50uM) 0.2uL 6.4uL 3.2uL 3.2uL
AP1V6 AP2V6 Mix (50uM) 0.2uL 6.4uL 3.2uL 3.2uL
Jan_09_CPG_D40809 (10uM) 0.2uL 6.4uL(5uL) 3.2uL 3.2uL
Econo Taq 50uL 1600uL 800uL 800uL


  • Mix content following the order from top to bottom in 2x 2 mL tube vortex and spin down
  • Aliquot 100uL using P200 pipette to strip 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

  • Skiped one cycle

File:20090501 CPGD40809.jpg

Step 2: Probe purification with ethanol precipitation

  • 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

Step 3: Purify the reaction with Qiaquick columns

  • Add 5X sample volume of PB buffer into each tube
  • 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 EB Buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
  • Repeat centrifuge
  • Measure concentration with Nanodrop

Step 4: Adapter Removal

Exonuclease treatment

  • To the 50ul Qiaquick purified amplicons
  • add 10ul 10X Lambda Exo Buffer, 5ul 10x Lambda exo
  • Incubate at37C 30min>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 EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
  • Repeat centrifuge
  • Measure concentration with Nanodrop

Incubation with USER enzyme:

  • Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.

Annealing of DpnII oligo and incubation with DpnII.

  • add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM)
  • Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold
  • Qiaquick column purification
  • Elute with 60uL EB Buffer