AlanFung:LabNotes/Probe/2009-4-23
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Step 4: Adapter Removal[edit]
Exonuclease treatment
Done on 4/22/09
- Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
- Add in (1/10 of total volume) 6uL 10X Exo buffer
- Incubate at37C 4h>90C 10m>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 ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
- 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
- Tube A (04/22/09-1) 260/280 1.84 7.9ng/uL
- Tube B (04/22/09-1) 260/280 1.78 8.5ng/uL
- Tube C (04/22/09-2) 260/280 1.66 8ng/uL
- Tube D (04/22/09-2) 260/280 1.76 6.4ng/uL
- Concentration too low, probably due to over digest by Lambda Exo , next time run 24 wells to one tube
- Combine Tube A(4/21) B(4/21) D(4/22-2) TUBE A
- Combine A(4/22-1) B((4/22-1) C(4/22-2) by qiaquick column TUBE B
- pool 3 tubes into one
- add in 750uL PB buffer mix by repeat pipetting
- load 450 mix to column
- Spin down @ 14,000 rpm 1m
- Discard flow through
- Load the remaining 450uL into column
- spin down @14,000 rpm 1m
- Add in 750ul PE wash buffer
- Spin down @ 14,000rpm 1m
- Discard flow through
- Repeat centrifuge
- Place into new collection tube
- Leave air dry for 5 m
- add in 50uL DDH2O
- Sit for 5m
- spin down at 14,000rpm for 1m
- Measure with Nanodrop
- Decided to restart experiment, with alice's primer and new tube of lambda exo
Step 1: PCR reaction system setup 48 wells[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 in the order of water>SYBR Green>Primer >template>econotaq
- Aliquot 105uL using P200 pipette to strip tubes on cooling rack, leave unused 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 File:20090423 CPGD40809.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 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
- Break pellet by repeat pipetting
- store in 4C overnight
Step 3: Purify the reaction with Qiaquick columns[edit]
- Add 5X sample volume of PB buffer into each tube (500uL)
- 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