AlanFung:LabNotes/Probe/2009-4-22: Difference between revisions
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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 | |||
{| border="1" align="center" | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''X24''' | |||
| align="center" style="background:#f0f0f0;"|'''X48''' | |||
| align="center" style="background:#f0f0f0;"|'''X72''' | |||
| align="center" style="background:#f0f0f0;"|'''X96''' | |||
| align="center" style="background:#f0f0f0;"|'''1.5 mL Tube''' | |||
|- | |||
|Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|9.6uL||align="right"|14.4uL||align="right"|19.2uL||align="right"|2.4uL | |||
|- | |||
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|650uL | |||
|- | |||
|AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL | |||
|- | |||
|RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|600uL | |||
|- | |||
|50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL | |||
|- | |||
|Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|4848uL||align="right"|7272uL||align="right"|9696uL||align="right"|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== | |||
*Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray | |||
{| border="1" align="center" | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume Needed''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
|100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL | |||
|- | |||
|3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL | |||
|- | |||
|Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL | |||
|- | |||
|Sample||align="right"|Sample Volume||align="right"|2400uL | |||
|- | |||
|Total Volume | |||
|colspan="2" align="right"|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 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 | |||
==Result== | |||
*PCR amplification relatively low compare to professor's reaction | |||
*Ethanol precipitation blue pellete very small | |||
==Suggestion== | |||
*Repeat experiment following professor's pcr condition (94C fopr 20S instead of 30S) | |||
==Reapeat Experiment== | |||
==Step 1: PCR reaction system setup== | ==Step 1: PCR reaction system setup== | ||
*Thaw Econo Taq on ice | *Thaw Econo Taq on ice |
Revision as of 22:19, 22 April 2009
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 | 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
- 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 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
Result
- PCR amplification relatively low compare to professor's reaction
- Ethanol precipitation blue pellete very small
Suggestion
- Repeat experiment following professor's pcr condition (94C fopr 20S instead of 30S)
Reapeat Experiment
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 | 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
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 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