AlanFung:LabNotes/Probe/2009-5-1
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Purpose
- To improve my prob preping skills
- Be extremely careful with contamination, clean every tools and equipments before use
- Following kun's protocol exactly the way he did it on 04/16/09
Step 1: PCR reaction system setup
- Thaw Econo Taq on ice
- Prepare 4 strips and 2x 2ml tubes on cooling rack
- Add in Content in table to tubes accordingly
Content | Volume | X32 | 2 mL Tube | 2 mL Tube | |
RNAse free H2O | 49uL | 1568uL | 784uL | 784uL | |
50X SYBG I | 0.4uL | 12.8uL | 6.4uL | 6.4uL | |
AP1V6 AP2V6 Mix (50uM) | 0.2uL | 6.4uL | 3.2uL | 3.2uL | |
AP1V6 AP2V6 Mix (50uM) | 0.2uL | 6.4uL | 3.2uL | 3.2uL | |
Jan_09_CPG_D40809 (10uM) | 0.2uL | 6.4uL(5uL) | 3.2uL | 3.2uL | |
Econo Taq | 50uL | 1600uL | 800uL | 800uL |
- Mix content following the order from top to bottom in 2x 2 mL tube vortex and spin down
- Aliquot 100uL 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) X16 > 72C 3M > 15C Hold
- Skiped one cycle
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
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 EB Buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
- Repeat centrifuge
- Measure concentration with Nanodrop
Step 4: Adapter Removal
Exonuclease treatment
- To the 50ul Qiaquick purified amplicons
- add 10ul 10X Lambda Exo Buffer, 5ul 10x Lambda exo
- Incubate at37C 30min>4C hold Saved as program Alan>EXOAR
Purify the reaction with Quaquick columns
- Add 5X sample volume of PB buffer into each tube, mix well
- 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 EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
- Repeat centrifuge
- Measure concentration with Nanodrop
Incubation with USER enzyme:
- Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.
Annealing of DpnII oligo and incubation with DpnII.
- add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM)
- Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold
- Qiaquick column purification
- Elute with 60uL EB Buffer