SXZ / mRNA ISOLATION PROTOCOL mRNA Isolation from total RNA with Oligotex mRNA mini Kit (Qiagen, Cat# 70022) Before starting: -Heat Oligotex Suspension to 37°C in a water bath or heating block. Mix by vortexing, and then place at room temperature. - Heat a water bath or heating block to 70°C, and heat Buffer OEB. -Bring the RNA samples (<250ug) to 250ul with RNase-free water -Add 250ul of Buffer OBB, mix well -Add 15ul of Oligotex suspension, mix well -Incubate the samples at 70C for 3min. -Incubate the samples at room temperature for 10-20min -Centrifuge at max. speed for 2 min - Remove the supernatant by pipetting -Re-suspend the Oligotex-RNA pellet in 400ul buffer OW2 by pipetting -Apply the Oligotex-RNA pellet to a spin colum -Centrifuge for 1 min at Max speed -Transfer the column to a new 1.5ml tub, add 400ul of OW2, and mix well by pipetting -Centrifuge for 1 min at Max speed -Transfer the column to a new 1.5ml tub, -Apply 120ul of OEB buffer (70C) to the column, pipette up and down 5-8 time to re- suspend the resin -Centrifuge for 1 min at max. speed, -Apply 120ul of OEB buffer (70C) again to the column, pipette up and down 5-8 time to re-suspend the resin -Centrifuge for 1 min at max. speed, -For the two elution of total 240ul mRNA, to do precipitation by adding: 1ul Linear Acrylamine (5ug/ul) 60ul 2M sodium acetate , pH 4.4 1000ul 100% ETOH 100ul H2O -total volume should be 1.5ml -Mix well and keep at -80C for overnight. -Centrifuge the precipitation solution at 4C with max speed for 30min -Washing the pellets with 70% ETOH -Centrifuge at 4C with max speed for 30min -Remove the residual ETOH -Dry the pellets by 10min -Re-suspend the pellets in 10ul RNase-free water. -the mRNA is ready for cDNA library construction.
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