AlanFung:LabNotes/Probe/2009-5-1
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Purpose[edit]
- 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[edit]
- 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[edit]
- Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content | Volume Needed | Volume |
100% Ethanol | 2.5X Sample Volume | 8000uL |
3M NaOAC | 0.1X Sample Volume | 320uL |
Glycoblue | 1/300X Sample Volume | 10.7uL |
Sample | Sample Volume | 3200uL |
Total Volume | 11530.7uL |
- Pool all 32 wells PCR product into tray using multichannel pipette(P300)
- Mix all content by repeat pipetting using 10mL Pipette
- Aliquot to 2 x 15mL tubes
- Store in -80C for more than 20 mins
- Turn on Centrifuge fix temperature 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
- Add in 350uL cold 75% Ethanol transfer pellet to 1.5mL tube
- Addin another 350uL 75% ethanol wash the 15ml tube and transfer 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 (500uL) of PB buffer into each tube
- Vortex and spin down
- 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 air dry
- Add in 50uL EB Buffer to each column let stand for 5min
- Repeat centrifuge
- Measure concentration with Nanodrop
- Tube A 260/280 OD: 1.95 108.3ng/uL * 50uL
- Tube B 260/280 OD: 1.96 122.5ng/uL * 50uL
Step 4: Adapter Removal[edit]
Exonuclease treatment In a PCR tube
- 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
- Airdry the column/tube assemblies
- Add in 50uL EB buffer to each column let stand for 5 min
- Repeat centrifuge
- Measure concentration with Nanodrop
- Tube A 260/280 OD: 1.84 8.6ng/uL * 50uL
- Tube B 260/280 OD: 1.93 15.9ng/uL * 50uL
Incubation with USER enzyme:
- Add 5 uL of USER (1K/ml)enzyme incubate for 30min on 37C.
- Take out 2uL from each tube for quantification by running a TBU gel
- PCR Tube A: 0.5uL 10bp ladder + 4.5uL ddh2o + 5uL 2X buffer
- PCR Tube B: 2uL sample + 2uL ddh2o + 4uL 2X buffer
- PCR Tube C 2uL sample + 2uL ddh2o + 4uL 2X buffer
- Pre-run gel for 30min, flush wells with P1000 and P200
- Denature all 3 tubes at 75C for 30min
- Transfer to cooling rack
- Load samples and ladder to gel (Ladder_Tube A_Tube B)
- Run gel for 30min at 200V
- Stain gel with 4uL sybr gold
- put on orbital shaker for 5 min
- Take image
File:ZhangLab 2 2009-05-01 16hr 23min.jpg
Annealing of DpnII oligo and incubation with DpnII.
- add 10ul of 10X DpnII buffer and 5uL RE-DpnIIs guide oligo (100uM)
- Perform 94C 2m > 0.1C/sec to 37C > add 5ul of DpnII restriction enzyme -> 37C 2 hours > 75C 20 min > 37 4 hours > 4C hold