AlanFung:LabNotes/Probe/2009-4-23
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Step 4: Adapter Removal
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
Load the remaining 450uL into column
- spin down @14,000 rpm 1m