AlanFung:LabNotes/Probe/2009-4-26

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Purpose[edit]

  • Rerun prop prep for CPG 100K with half plate (48wells)
  • Try to figure out the low yield problem after Lamda Exo digestion

Strategy[edit]

  • All purification elute with EB buffer instead of water
  • Omit the enzyme inactivation of Lambda Exo by high temperature, remove enzyme by purification using qiaquick column instead

Procedures[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 in the order of water>SYBR Green>Primer >template>econotaq
  • 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) X16 > 72C 3M > 15C Hold File:20090426 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 12,000uL
3M NaOAC 0.1X Sample Volume 480uL
Glycoblue 1/300X Sample Volume 16uL
Sample Sample Volume 4800uL
Total Volume 17,296uL
  • Pool all 48 wells PCR product into tray using multichannel pipette(P300)
  • Mix all content by repeat pipetting using 10mL pipette
  • Aliquot mixture 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[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 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[edit]

Exonuclease treatment

  • Add in 3uL Lambda Exonuclease (5K/mL)
  • Add in (1/10 of total volume) 6uL 10X Exo buffer
  • Incubate at 37C 1h

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 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 50uL EB Buffer

Result[edit]

  • Samples seem to be over amplified running 17 cycles, contradicting the data form last run. Observe the process next time
  • Solved the low yield problem
  • Continue on 04/27/09


After Ethanol Precipitation

  • TUBE A 260/280 1.95 171 ng/uL*50uL
  • TUBE B 260/280 1.96 177.7 ng/uL*50uL


After Lambda Exo Incubation

  • TUBE A 260/280 1.91 26.8 ng/uL*50uL
  • TUBE B 260/280 2.00 30.8 ng/uL*50uL


After Adaptor Removal

  • TUBE A 260/280 2.18 14.1 ng/uL*50uL
  • TUBE B 260/280 2.07 15.7 ng/uL*50uL