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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 *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
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