AlanFung:LabNotes/Probe/2009-4-12
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Production PCR for probe CPG_D40809 (24 wells)[edit]
Objective[edit]
- 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[edit]
- 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[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
Procedure[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 | 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[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
Results[edit]
- 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[edit]
- Set only 18 cycles
- Observe at cycle 16 skip to next step once drop started