AlanFung:LabNotes/Probe/2009-5-1

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Purpose[edit]

  • 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[edit]

  • 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[edit]

  • Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content Volume Needed Volume
100% Ethanol 2.5X Sample Volume 8000uL
3M NaOAC 0.1X Sample Volume 320uL
Glycoblue 1/300X Sample Volume 10.7uL
Sample Sample Volume 3200uL
Total Volume 11530.7uL
  • Pool all 32 wells PCR product into tray using multichannel pipette(P300)
  • Mix all content by repeat pipetting using 10mL Pipette
  • Aliquot to 2 x 15mL tubes
  • Store in -80C for more than 20 mins
  • Turn on Centrifuge fix temperature at 4C
  • 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 350uL cold 75% Ethanol transfer pellet to 1.5mL tube
  • Addin another 350uL 75% ethanol wash the 15ml tube and transfer to 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[edit]

  • Add 5X sample volume (500uL) of PB buffer into each tube
  • Vortex and spin down
  • 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 air dry
  • Add in 50uL EB Buffer to each column let stand for 5min
  • Repeat centrifuge
  • Measure concentration with Nanodrop
  • Tube A 260/280 OD: 1.95 108.3ng/uL * 50uL
  • Tube B 260/280 OD: 1.96 122.5ng/uL * 50uL

Step 4: Adapter Removal[edit]

Exonuclease treatment In a PCR tube

  • 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
  • Airdry the column/tube assemblies
  • Add in 50uL EB buffer to each column let stand for 5 min
  • Repeat centrifuge
  • Measure concentration with Nanodrop
  • Tube A 260/280 OD: 1.84 8.6ng/uL * 50uL
  • Tube B 260/280 OD: 1.93 15.9ng/uL * 50uL

Incubation with USER enzyme:

  • Add 5 uL of USER (1K/ml)enzyme incubate for 30min on 37C.
  • Take out 2uL from each tube for quantification by running a TBU gel
  • PCR Tube A: 0.5uL 10bp ladder + 4.5uL ddh2o + 5uL 2X buffer
  • PCR Tube B: 2uL sample + 2uL ddh2o + 4uL 2X buffer
  • PCR Tube C 2uL sample + 2uL ddh2o + 4uL 2X buffer
  • Pre-run gel for 30min, flush wells with P1000 and P200
  • Denature all 3 tubes at 75C for 30min
  • Transfer to cooling rack
  • Load samples and ladder to gel (Ladder_Tube A_Tube B)
  • Run gel for 30min at 200V
  • Stain gel with 4uL sybr gold
  • put on orbital shaker for 5 min
  • Take image

File:ZhangLab 2 2009-05-01 16hr 23min.jpg


Annealing of DpnII oligo and incubation with DpnII.

  • add 10ul of 10X DpnII buffer and 5uL RE-DpnIIs guide oligo (100uM)
  • Perform 94C 2m > 0.1C/sec to 37C > add 5ul of DpnII restriction enzyme -> 37C 2 hours > 75C 20 min > 37 4 hours > 4C hold