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]
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[edit]
- Set only 18 cycles
- Observe at cycle 16 skip to next step once drop started