AlanFung:LabNotes/Probe Synthesis Nicking/2009-5-5

From ZhangLabWiki
Revision as of 19:29, 8 May 2009 by >Alan6017518 (→‎Step 1: PCR reaction system setup (48wells))
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Overview[edit]

  • PCR reaction system setup
  • Probe purification with ethanol precipitation
  • Purify the reaction with Qiaquick columns
  • Adapter Removal
  • Page Denaturing Gel
  • Purify the product from the gel
  • Precipitation
  • Quantification of DNA using denaturing gel and a quantitative ladder

Procedures[edit]

Step 1: PCR reaction system setup (48wells)[edit]

  • Thaw 2 tubes of 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
BCM3 (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

  • Skip at cycle 14

File:20090505 Nicking110154.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 12mL
3M NaOAC 0.1X Sample Volume 480uL
Glycoblue 1/300X Sample Volume 16uL
Sample Sample Volume 4800uL
Total Volume 17296uL
  • Pool all 48 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 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
  • Gel Run

File:ZhangLab 2 2009-05-06 09hr 52min crop.jpg

Step 4: Adapter Removal[edit]

File:ZhangLab 2 2009-05-06 09hr 52min crop.jpg Exonuclease treatment

  • 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 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
  • Add 40ul ddH2O, 5ul USER, 37C 1h


  • Add 10ul 10x DpnII buffer, 5ul RE-DpnII-ns2 guide oligo,

94C 2min -> 37C 3min -> add 5ul DpnII -> 37C 6h -> 75C 20min -> 4c hold Purified with Qiaquick

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