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DNase digestion Protocol

互联网2013-11-13

2464

实验步骤

1. Follow the standard protocol until the samples completely pass through the HiBind® RNA Minicolumn (Steps 1-4). Prepare the following:

1) Add 300ul of RNA wash Buffer I to the column and centrifuge at .10,000 x G for 1 minute

2) For each HiBind® RNA Minicolumn, prepare the DNase I digestion reaction mix as follows:

OBI DNase I Digestion Buffer 73.5ul

RNase-free DNase I (20 Kunitz unites/ul) 1.5ul

Total volume 75ul

a. DNase I is very sensitive and prone to physical denaturing; so do not vortex the DNase I mixture. Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before RNA isolation.

b. OBI DNase I digestion buffer is supplied with OBI RNase-free Dnase set.

c. Standard DNase buffers are not compatible with on-membrane Dnase digestion.

3) Pipet 75ul of the DNase I digestion reaction mix directly onto the surface of the HiBind® RNA resin in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion will not be complete if some of the mix sticks to the wall or the O-ring of the HiBind® RNA Mini column.

4) Incubate at room temperature(25-30°C) for 15 minutes

2. Place column in a clean 2ml collection tube, and add 500ul RNA Wash Buffer I. Incubate 5 minutes at room temperature. Centrifuge and discard flow-through. Reuse the collection tube.

3. Place column in the same 2ml collection tube, and add 500ul RNA Wash Buffer II diluted with ethanol. Centrifuge and discard flow-through. Reuse the collection tube.

Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

4. Wash column with a second 500ul of Wash Buffer II by repeating step 3. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 1 min at full speed to completely dry the HiBind® matrix.

5. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 50-100ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution may be necessary if the expected yield of RNA >50ug. Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 70°C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.

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