AlanFung:LabNotes/Probe/2009-4-12

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Revision as of 21:05, 17 April 2009 by >Alan6017518 (→‎Procedure)
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Production PCR for probe CPG_D40809 (24 wells)

Objective

  • Get familiar with the probe synthesis procedure
  • Optimization of Padlock Probe Preparation
  • Repeat PCR reaction setup and Probe purification with ethanol precipitation with optimized condition

Samples & Materials

  • Jan_09_CPG_D40809 100K Oligo (10uM)
  • Econo Taq
  • AP1V6 AP2V6 Primer Mix (50uM)
  • RNAse free H2O
  • 50X SYBG I
  • 3M NaOAC
  • GlycoBlue
  • 100% Ethanol

Overview

  • 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

Procedure

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4.8

9.6

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4800

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9.6

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38.4

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Step 1: PCR reaction system setup

  • Thaw Econo Taq on ice
  • Prepare strips and tubes on cooling rack
  • Add in Content in table to tubes accordinly
Content Volume X24 2.0mL Tube A 2.0mLTube B
Jan_09_CPG_D40809 (10uM) 0.2uL 4.8uL 2.4uL 2.4uL
Econo Taq 50uL 1250uL 626uL 625uL
AP1V6 AP2V6 Mix (50uM) 0.4uL 9.6uL 4.8uL 4.8uL
RNAse free H2O 50uL 1200uL 600uL 600uL
50X SYBG I 0.4uL 9.6uL 4.8uL 4.8uL
Total Volume 101uL 2424uL 1212uL 1212uL
  • Mix content in 2 x 2.0mL tube vortex and spin down
  • Aliquot 100uL using P200 pipette to 24 wells (3 strips) on cooling rack
  • Seal strips with strip caps
  • Place into Real Time PCR
  • Perform

94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X17 > 72C 3M > 15C Hold

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

Results

File:20090412 CPGD40809.jpg

  • Using P200 to aliquot gives a much more constant amplification throughout all the 24 wells
  • P200 also minimized the lost of solution during the aliquot compare to the repeater
  • Samples over amplified, due to SYBR Green binding to single stranded DNA.

Suggestion

  • Set only 18 cycles
  • Observe at cycle 16 skip to next step once drop started