Protocol for mRNA amplification，非常详细的RT-PCR实验过程[Howard Hughes Medical
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	b-ME
80 ul	H20
350 ul	RLT

Add 100uL of RLT to in-vitro transcription tube and mix well.

Transfer contents of in vitro transcription mix to 1.5mL RNase/DNase-free tube.

Add 330 uL of RLT mix. (stopping point –80 overnight)

Add 250ul 95% Ethanol and mix well by pipetting. (Do not spin here!)

Apply sample (700ul) to RNeasy mini spin column sitting in a collection tube.

Centrifuge 15 sec at >= 8000 x g. Discard flow through.

Protocol for mRNA amplification

aRNA purification using Qiagen RNeasy COLUMNS (cont’d)

Transfer RNeasy column to a new 2-ml collection tube (supplied).

Add 500ul Buffer RPE (which must contain ethanol) and centrifuge 15 sec at >=8000 x g.

Discard flow-through but re-use tube.

Add another 500ul Buffer RPE to the RNeasy column.

Centrifuge for 2 min at maximum speed.

Discard flow-through but re-use tube

Add another 500ul Buffer RPE to the RNeasy column.

Centrifuge for 2 min at maximum speed.

Place RNeasy spin column into a new 1.5-ml collection tube (supplied).

Centrifuge for 1 min at maximum speed to completely dry column.

Transfer RNeasy column into a new RNase, DNase-free 1.5-ml collection tube (not supplied).

Add 30ul RNase-free water directly onto membrane.

Centrifuge for 1 min at >=8000 x g to elute.

Repeat if expected yield is >= 30ug.

Check RNA concentration and quality by measuring OD260 and OD260/280.

Check RNA quality by running a gel.

Add 1uL of Sample to 1-2uL of loading dye.

Incubate at 65C for 10min. Put on ice immediately after incubation.

Run on a 1.25% agarose gel (2.5g agarose in 200mL 1X MOPS) in 1X MOPS Buffer for 45 minutes to 1.5hr at 165 V.

second round amplification

In PCR reaction tube, mix