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
- 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) X15 > 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 one tube in -80C for 20 mins
- Store the other tube on ice for 20 mins
- Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
- Centrifuge frozen 15mL tubes at 3000rpm for 30m 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
File:20090422 CPGD40809.jpg
- PCR amplification relatively low compare to professor's reaction
File:Kun 20090416 production-CpgJan09-repeat-PCR.jpg
- Ethanol precipitation blue pellete very small
- No difference observed between incubation temperature at 0C and -80C
Suggestion[edit]
- Repeat experiment following professor's pcr condition (94C fopr 20S instead of 30S)
Repeat Experiment[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
- 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 20S > 58C 30S > 72C 30S>Plate Read) X15 > 72C 3M > 15C Hold
File:20090422 2 150253.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 |
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 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 500uL cold 75% Ethanol, transfer pellet to a 1.5mL tube
- Wash 15mL tube with 250mL cold 75% Ethanol, trasnfer all ethanol to 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 ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
- Repeat centrifuge
- Measure concentration with Nanodrop
- Tube A (04/22/09-1) 260/280 1.9 81.4ng/uL
- Tube B (04/22/09-1) 260/280 1.81 74.4ng/uL
- Tube C (04/22/09-2) 260/280 1.85 73.1ng/uL
- Tube D (04/22/09-2) 260/280 1.88 69.63ng/uL