AlanFung:LabNotes/Probe/2009-4-22: Difference between revisions
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*Mix all content by repeat pipetting using multichannel pipette | *Mix all content by repeat pipetting using multichannel pipette | ||
*Aliquot about 3750uL to 2 x 15mL tube | *Aliquot about 3750uL to 2 x 15mL tube | ||
*Store in -80C for | *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 | *Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge | ||
*Centrifuge frozen 15mL tubes at 3000rpm for | *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''' | *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 | *Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube | ||
Line 71: | Line 72: | ||
[[Image:Kun 20090416_production-CpgJan09-repeat-PCR.jpg]] | [[Image:Kun 20090416_production-CpgJan09-repeat-PCR.jpg]] | ||
*Ethanol precipitation blue pellete very small | *Ethanol precipitation blue pellete very small | ||
*No difference observed between incubation temperature at 0C and -80C | |||
==Suggestion== | ==Suggestion== | ||
Line 112: | Line 114: | ||
*Perform | *Perform | ||
94C 2M > ( '''94C 20S''' > 58C 30S > 72C 30S>Plate Read) X15 > 72C 3M > 15C Hold | 94C 2M > ( '''94C 20S''' > 58C 30S > 72C 30S>Plate Read) X15 > 72C 3M > 15C Hold | ||
[[Image:20090422_2_150253.jpg]] | |||
==Step 2: Probe purification with ethanol precipitation== | ==Step 2: Probe purification with ethanol precipitation== | ||
Line 140: | Line 143: | ||
*Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C | *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''' | *Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' | ||
*Add in | *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 | *Centrifuge at 10,000rpm for 5min at 4C | ||
*Remove all liquid, air dry sample for 5min or until samples dried out | *Remove all liquid, air dry sample for 5min or until samples dried out | ||
*Resuspend the DNA with 100uL RNAse free H2O | *Resuspend the DNA with 100uL RNAse free H2O | ||
==Step 3: Purify the reaction with Qiaquick columns== | |||
*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''' |
Latest revision as of 01:40, 23 April 2009
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
Result[edit]
- 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