AllPrep RNA/DNA Extraction Protocol
TASC
Joanna Liliental 1/30/2012
DNA
THE FLOW THROUGH IS RNA
The membrane binds gDNA.
RNA
BEFORE STARTING:
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Add 10ul beta mercaptoethanol to 1ml RLT Plus Buffer. (can store at RT for up to 1 month).
Add 4 vol of EtOH to Buffer RPE.
Label tubes.
AllPrep RNA/DNA Extraction Protocol
TASC
Joanna Liliental 1/30/2012
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Add 350ul (up to 1x106 cells) to the cell pellet.
Pipet up and down and leave on ice for 5-10 min.
Centrifuge lysate for 3 min at max speed.
Transfer supe to DNA SPIN COLUMN placed in a 2ml collection tube.
Close lid gently
Centrufuge 30sec @ 10,000rpm.
FLOW THROUGH  use for RNA purification
7. Place DNA SPIN COLUMN in a new 2ml collection tube.
8. Store at RT or +4C for later DNA purification. (NO FREEZING!!!) or PROCEED WITH gDNA
PROTOCOL..
RNA
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Add 1 volume of 70% EtOH to flow-through form #7 above.
Mix well by pipetting.
Proceed immediately to next step.
Transfer sample (up to 700ul), including any ppt, to an RNeasy SPIN COLUMN placed in a 2 ml
collection tube.
Centrifuge for 15 sec @10,000rpm.
Discard flow-through.
Add 700ul Buffer RW1 to RNeasy SPIN COLUMN.
Close lid.
Centrifuge for 15 sec @ 10,000rpm.
Discard flow-thorough.
Add 500ul Buffer RPE to RNeasy SPIN COLUMN.
Close lid.
Centrifuge for 15 sec @ 10,000rpm.
Discard flow-through.
Add 500ul Buffer RPE to Rneasy SPIN COLUMN.
Close lid.
Centrifuge 2 min @ 10,000rpm
Place Rneasy SPIN COLUMN in a new 2 ml collection tube.
Centrifuge 1 min @10,000 rpm to dry the membrane.
Place RNeasy SPIN COLUMN in a new 1.5 ml collection tube.
Add 30-50ul RNAse – free H2O.
Close the lid.
Centrifuge 1 min @ 10,000 rpm to elute RNA
AllPrep RNA/DNA Extraction Protocol
TASC
Joanna Liliental 1/30/2012
gDNA
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Add 500ul Buffer AW1 to DNA SPIN COLUMN (ina 2ml collection tube).
Close lid.
Centrifuge for 15 sec @ 10,000 rpm to WASH membrane.
Discard flow-through.
Add 500ul Buffer AW2 to DNA SPIN COLUMN.
Close lid.
Centrifuge 2 min @ 10,000 rpm to WASH membrane.
Place SPIN COLUMN in a new 1.5ml collection tube.
Add 100ul Buffer EB directly to the spin column membrane.
Close lid.
Incubate at RT 1min.
Centrifuge 1 min @ 10,000 rpm to ELUTE gDNA.