AlanFung:LabNotes/Probe/2009-4-22

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Step 1: PCR reaction system setup[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
  • Aliquot 105uL 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) X15 > 72C 3M > 15C Hold

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 one tube in -80C for 20 mins
  • Store the other tube on ice for 20 mins
  • Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
  • Centrifuge frozen 15mL tubes at 3000rpm for 30m 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

Result[edit]

File:20090422 CPGD40809.jpg

  • PCR amplification relatively low compare to professor's reaction

File:Kun 20090416 production-CpgJan09-repeat-PCR.jpg

  • Ethanol precipitation blue pellete very small
  • No difference observed between incubation temperature at 0C and -80C

Suggestion[edit]

  • Repeat experiment following professor's pcr condition (94C fopr 20S instead of 30S)


Repeat Experiment[edit]

Step 1: PCR reaction system setup[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
  • Aliquot 105uL using P200 pipette to strip tubes on cooling rack
  • Seal strips with strip caps
  • Place into Real Time PCR
  • Perform

94C 2M > ( 94C 20S > 58C 30S > 72C 30S>Plate Read) X15 > 72C 3M > 15C Hold File:20090422 2 150253.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 500uL cold 75% Ethanol, transfer pellet to a 1.5mL tube
  • Wash 15mL tube with 250mL cold 75% Ethanol, trasnfer all ethanol 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 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 ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
  • 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