AlanFung:LabNotes/Probe/2009-4-26
Jump to navigation
Jump to search
Purpose[edit]
- Rerun prop prep for CPG 100K with half plate (48wells)
- Try to figure out the low yield problem after Lamda Exo digestion
Strategy[edit]
- All purification elute with EB buffer instead of water
- Omit the enzyme inactivation of Lambda Exo by high temperature, remove enzyme by purification using qiaquick column instead
Procedures[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 in the order of water>SYBR Green>Primer >template>econotaq
- 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) X16 > 72C 3M > 15C Hold File:20090426 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 | 12,000uL |
3M NaOAC | 0.1X Sample Volume | 480uL |
Glycoblue | 1/300X Sample Volume | 16uL |
Sample | Sample Volume | 4800uL |
Total Volume | 17,296uL |
- Pool all 48 wells PCR product into tray using multichannel pipette(P300)
- Mix all content by repeat pipetting using 10mL pipette
- Aliquot mixture 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[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 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[edit]
Exonuclease treatment
- Add in 3uL Lambda Exonuclease (5K/mL)
- Add in (1/10 of total volume) 6uL 10X Exo buffer
- Incubate at 37C 1h
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 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 50uL EB Buffer
Result[edit]
- Samples seem to be over amplified running 17 cycles, contradicting the data form last run. Observe the process next time
- Solved the low yield problem
- Continue on 04/27/09
After Ethanol Precipitation
- TUBE A 260/280 1.95 171 ng/uL*50uL
- TUBE B 260/280 1.96 177.7 ng/uL*50uL
After Lambda Exo Incubation
- TUBE A 260/280 1.91 26.8 ng/uL*50uL
- TUBE B 260/280 2.00 30.8 ng/uL*50uL
After Adaptor Removal
- TUBE A 260/280 2.18 14.1 ng/uL*50uL
- TUBE B 260/280 2.07 15.7 ng/uL*50uL