Important Notes:

Brief procedure:

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Specification:

v 0317

FavorPrep 96-Well Total RNA Kit- For isolation of total RNA from aminal cells or tissues

TM

(For Research Use Only)

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Quality ControlThe quality of 96-Well Total RNA Kit is tested on a lot-to-lot basis. The purified RNA is checked byagarose gel analysis and quantified with spectrophotometer.

centrifuge protocol vacuum protocol

∙ Transfer the sample mixture to combined plate. (Filter Plate + third 96-well, 2 ml Plate) ∙ Centrifuge and discard the flow-through.

● STEP 4. Bind RNA to Filter Plate:

∙ Transfer the sample mixture to a Filter Plate assembled with manifold and third 96-well, 2 ml Plate . ∙ Apply vacuum until the wells have emptied and discard the flow -through.

Kit Contents:FATRE 96002 (2 plates)

FATRE 96004 (4 plates)

FATRE 96001 (1 plate)Cat. No.:

Lysis Buffer 60 ml 120 ml 120 ml x 2Wash Buffer 1 * (concentrate) 44 ml 88 ml 88 ml x 2Wash Buffer 2 ▀ (concentrate) 25 ml 50 ml 50 ml x 2RNase-Free Water 15 ml 30 ml 30 ml x 2Filter Plate (96-Well RNA plate) 1 plate 2 plates 4 plates96-Well 2 ml Plate 4 plates 8 plates 16 platesElution Plate (96-Well PCR plate) 1 plate 2 plates 4 platesAdhesive Film 2 pcs 4 pcs 8 plates

Seal with Adhesive Film.

Collect samples in thefirst 96-well, 2 ml plate

Centrifuge at 5,600 – 6,000 x g, 10 min

● STEP 5. Wash Filter Plate with Wash Buffer 1 ∙ Add Wash Buffer 1. ∙ Apply vacuum for and discard the flow-through.

∙ Add Wash Buffer 1.∙ Centrifuge and discard the flow -through.

∙ Stand the Filter plate on a clean paper towel at RT for 10 min.

● STEP 8. Dry the membranes of Filter Plate:

● STEP 9. RNA Elution:∙ Add RNase-free Water to the Filter Plate. Stand for 3 min. ∙ Centrifuge to elute RNA.

．Add RNase-free Water to the Filter Plate. Stand for 3 min. ．Apply vacuum to elute RNA.

Material to be provided by user for a 96-well preparation1. Centrifuge equiment with a swing -bucket rotor and adaptor for 96-well plate, capable of at least 5,600 ~ 6,000 X g.2. Vacuun manifold for 96-well plate and a vaccum source at 10 inches Hg.3. ß-mercaptoethanol (ß-Me)3. 96 ~100 % ethanol

→

→ →

Filter Plate

→ →

the third96-well,2 ml plate

transfer the sample mixtureto the Filter Plate

Vaccum Monifold

add Wash Buffer 1 andapply vacuum at 10 inches Hg

→add WashBuffer 1

centrifuge,4,500 – 6,000 x g,2 min.

centrifuge,4,500 – 6,000 x g,2 min.

∙ Add Wash Buffer 2. ∙ Apply vacuum for and discard the flow-through.

∙ Add Wash Buffer 2.∙ Centrifuge and discard the flow -through.

Vaccum Monifold

STEP 6 : add Wash Buffer 2 and apply vacuumat 10 inches Hg for until the wells have emptied.STEP 7: add Wash Buffer 2 and apply vacuumat 10 inches Hg 10 min.

→STEP 6: Add Wash Buffer 2 and centrifuge 5,600 - 6,000 x g, 2 min STEP 7: Add Wash Buffer 2 and centrifuge 5,600 - 6,000 x g, 15 min

Vaccum Monifold

Filter Plate

Vaccum MonifoldVaccum Monifold

→ →

the third96-well,2 ml plate

vacuum manifold

transfer the samplemixture to the Filter Plate and apply vacuumat 10 inches Hg.

Manifold lid

Add RNase-free Water to the Filter Plateand applyvacuum at 10 inches Hgfor 10 min

PCR Plate

Manifold lid

the fourth96-well,2 ml plate

Vaccum Monifold

Filter Plate

Vaccum Monifold

Vaccum Monifold

→

．Gently tap the tips of the Filter Plate on paper towel to remove residual liquid．Apply maximum vacuum for an addition 10 minutes.

Add RNase-free ethanol (96~100%) to Wash Buffer 1 and Wash Buffer 2 when first use.

* Ethanol volume for Wash Buffer 1▀ Ethanol volume for Wash Buffer 2

FATRE 96001 8 ml

FATRE 96002 FATRK 96004 16 ml

200 ml 100 ml

Principle: Filter Plate (silica membrane)Sample size: up to 1 x 10 animal cells or 50 mg tissues / preparationProcessing: centrifugation protocol or vacuum & centrifugation protocol Operation time: < within 60 min/ 96 preparationRNA Binding capacity: up to 75 µg/ wellElution volume: 50 ~ 75 µl

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1. Make sure everything is RNase-free when handling RNA.2. Buffers provided in this system contain irritants. Wear gloves and lab coat when andling these buffers.3. Caution: ß-mercaptoethanol (ß-Me) is hazardous to human health. perform the procedures involving ß-M in a chemical fume hood. 4. Add RNase-free ethanol (96~100%) to Wash Buffer 1 and Wash Buffer 2 when first use.5. Prepare RNase-free DNase 1 reaction buffer (1M NaCl, 10 mM MnCl2, 20 mM Tris-HCl, pH 7.0 at 25ºC) and make the final concentration of DNase I to 0.5 U/ μl.

● STEP 1. Sample preparation and lysis

● STEP 2. Clarify lysate

● STEP 6 & 7. Wash Filter Plate with Wash Buffer 2

● STEP 3. Adjust binding condition:

or

→→

→Transfer upper clarifiedlysate to the second 96-well, 2ml plate

Add Lysis Buffer Disrupt the samples → Stand at room temperature, 5 min

Add 70 % ethanol Mix by pipetting→

Centrifuge protocol Vacuum protocol

● (Optional) : Digest DNA by DNase I

A1. Add Wash Buffer 1. Centrifuge for 5 min and discard the flow-through. A2. Add DNase I mixture. Stand at R.T for 15 min.A3. Add Wash Buffer 1. Centrifuge for 2 min and discard the flow-through. A4. After DNase I digestion, proceed to STEP 6.

B1. Add Wash Buffer 1. Apply vacuum for 2 min and discard the flow-through. B2. Add DNase I mixture. Stand at R.T for 15 min.B3. Add Wash Buffer 1. Apply vacuum until the wells have emptied and discard the flow-through. B4. After DNase I digestion, proceed to STEP 6.

centrifuge,4,500 – 6,000 x g,5 min.

Filter Plate

PCR Plate

the fourth96-well,2 ml plate

Add RNase-free Water to the Filter Plate

→

Vaccum Monifold

apply vacuum,10 inches Hg, 2 min

apply vacuum,10 inches Hg

Vaccum Monifold

→ →

add Wash Buffer 1 add DNase I, RT, 15 min

→

add Wash Buffer 1

proceed to STEP 6

→ →

add Wash Buffer 1 add DNase I, RT, 15 min

centrifuge at 4,500 – 6,000 x g,5 min.

centrifuge at 4,500 – 6,000 x g,2 min.

→

add Wash Buffer 1

→ proceed to STEP 6

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Protocol: (centrifugation processing)- using centrifuging force to handle DNA binding step and washing steps.

Protocol: (Vacuum processing)- using vacuum force to handle DNA binding step and washing steps.

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STEP 4. RNA Binding ∙ Place a Filter Plate (96-Well RNA Plate) on a clean 96-Well 2 ml Plate (provided, the third 96-well, 2 ml plate). ∙ Transfer the sample mixture (ethanol mixed) to each well of the Filter Plate. ∙ Place the combined plate (Filter Plate combined with the third 96-Well, 2 ml plate) in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 2 min. ∙ Discard the flow-through and return the Filter Plate to the third 96-Well 2 ml Plate.

STEP 5. Wash Filter Plate with Wash Buffer 1 ∙ Add 400 µl of Wash Buffer 1 (ethanol added) to each well of the Filter Plate. ∙ Place the combined plate (Filter Plate combined with the third 96-Well, 2 ml plate) in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 2 min. Discard the flow-through and return the Filter Plate to the third 96-Well, 2 ml Plate.STEP 6. Wash Filter Plate with Wash Buffer 2 ∙ Add 500 µl of Wash Buffer 2 (ethanol added) to each well of the Filter Plate. ∙ Place the combined plate (Filter Plate combined with the third 96-Well, 2 ml plate) in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 2 min. Discard the flow-through and return the Filter Plate to the third 96-Well, 2 ml Plate.

STEP 7. Repeat STEP 6. ∙ Wash the Filter Plate one more with Wash Buffer 2 ∙ Centrifuge at 5,600 – 6,000 x g for 15 min. Discard the flow-through.

STEP 3. Adjust binding condition ∙ Transfer 350 µl of the upper clarified lysate to each well of a clean 96-well, 2 ml plate (provided, the second 96-well, 2 ml plate). -- Note: Avoid to pipet any debris and pellet when transfering the supernatant. ∙ Add 350 µl of 70 % RNase-free ethanol to each well and mix by pipetting. -- Note: make sure that ethanol have been mixed completely.

STEP 2. Clarify lysate ∙ Seal the Adhesive Film on the first 96-well 2 ml plate. Place the plate in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 10 min.

STEP 8. Dry the membranes of Filter Plate ∙ Place the Filter Plate on top of a clean paper towel (not provided) and stand at room temperature for 10 minSTEP 9. RNA Elution ∙ Place a Elution Plate (provided, 96-Well PCR plate) on top of a clean 96-Well, 2 ml Plate (provided, the fourth 96-well, 2 ml plate) and place the Filter Plate on the Elution plate. (top: Filter Plate, middle: 96-well PCR Plate, bottom: 96-Well 2 ml plate) ∙ Add 50 ~ 75 μl of RNase-free Water to the membrane center of the Filter Plate. Stand for 3 min. ∙ Place the combined plate in a rotor bucket and centrifuge for 5 min at 5,600 – 6,000 x g for 5 min to elute RNA. ∙ Seal the Adhesive Film and store the RNA at -70 °C.

Please Read Important Notes Before Starting The Following Steps.

STEP 1. Sample preparation and lysis For animal cells: ∙ Transfer up to 1 x 10 cells to each well of a first 96-well 2 ml Plate. Centrifuge the plate at 500 x g, 4 °C for 5 min. Remove the supernatant. ∙ Add 450 µl of Lysis Buffer and 4.5 µl of ß-Mercaptoethanol. Pipet up and down to resuspend the cells completely. ∙ Incubate the sample mixture at room temperature for 5 min. For animal tissues : ∙ Transfer up 50 mg tissue to each well of a first 96-well 2 ml Plate. ∙ Add 450 µl of Lysis Buffer and 4.5 µl of ß-Mercaptoethanol. ∙ Disrupt the sample with a appropriate homogenizer. ∙ Incubate the sample mixture at room temperature for 5 min.

Material to be provided by user for a 96-well preparation

1. Centrifuge equiment with a swing -bucket rotor and adaptor for 96-well plate, capable of at least 5,600 ~ 6,000 X g.2. β- mercaptoethanol3. 96 ~100 % ethanol

Please Read Important Notes Before Starting The Following Steps.

1. Centrifuge equiment with a swing -bucket rotor and adaptor for 96-well plate, capable of at least 5,600 ~ 6,000 X g.2. Vacuun manifold for 96-well plate and a vaccum source.3. β- mercaptoethanol4. 70 % ethanol

Material to be provided by user for a 96-well preparation

STEP 3. Adjust binding condition ．Transfer 350 µl of the upper clarified lysate to each well of a clean 96-well, 2 ml plate (provided, the second 96-well, 2 ml plate). -- Note: Avoid to pipet any debris and pellet when transfering the supernatant. ．Add 350 µl of 70 % RNase-free ethanol to each well and mix by pipetting. -- Note: make sure that ethanol have been mixed completely.

STEP 2. Clarify lysate ．Seal the Adhesive Film on the first 96-well 2 ml plate. Place the plate in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 10 min.

STEP 1. Sample preparation and lysis For animal cells: ∙ Transfer up to 1 x 10 cells to each well of a first 96-well 2 ml Plate. Centrifuge the plate at 500 x g, 4 °C for 5 min. Remove the supernatant. ∙ Add 450 µl of Lysis Buffer and 4.5 µl of ß-Mercaptoethanol. Pipet up and down to resuspend the cells completely. ∙ Incubate the sample mixture at room temperature for 5 min. For animal tissues : ∙ Transfer up 50 mg tissue to each well of a first 96-well 2 ml Plate. ∙ Add 450 µl of Lysis Buffer and 4.5 µl of ß-Mercaptoethanol. ∙ Disrupt the sample with a appropriate homogenizer. ∙ Incubate the sample mixture at room temperature for 5 min.

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•(Optional STEP : Digest DNA by DNase I) Follow the steps from A1 ~ A4 to eliminate DNA. Otherwise, proceed to STEP 5 directly. •A1. Add 200 µl of Wash Buffer 1 (ethanol added) to each well of the Filter Plate. Place the combined plate in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 5 min. Discard the flow-through and return the Filter Plate to the third 96-Well 2 ml Plate. •A2. Add 50 µl of RNase-free DNase I solution (0.5U/ul, not provided) to each well’s membrane of the Filter Plate. Stand the plate for 15 min at room temperature. •A3. Add 200 µl of Wash Buffer 1 to the to each well of the Filter Plate. Place the combined plate in a rotor bucket and centrifuge at 5,600 – 6,000 x g for 2 min. Discard the flow-through and return the Filter Plate to the third 96-Well 2 ml Plate. •A4. After DNase I treatment, proceed to STEP 6.

STEP 4. RNA Binding ∙ Place a clean 96-well, 2 ml plate (provided, the third 96-well, 2 ml plate) on the rack of vacuum manifold and cover the manifold lid. Place a Filter Plate (96-well RNA Plate, provided) on top of the thrid 96-Well 2 ml plate. ∙ Transfer the sample mixture to the Filter Plate. ∙ Apply vacuum at 10 inches Hg until the wells have emptied. Discard the flow-through and return the Filter Plate to the manifold.

STEP 5. Wash Filter Plate with Wash Buffer 1 ∙ Add 400 µl of Wash Buffer 1 (ethanol added) to each well of the Filter Plate. ∙ Apply vacuum at 10 inches Hg until the wells have emptied. Discard the flow-through and return the Filter Plate to the manifold.

STEP 6. Wash Filter Plate with Wash Buffer 2 ∙ Add 500 µl of Wash Buffer 2 (ethanol added) to each well of the Filter Plate. ∙ Apply vacuum at 10 inches Hg until the wells have emptied. Discard the flow-through and return the Filter Plate to the manifold.

STEP 7. Repeat STEP 6. ∙ Wash the Filter Plate one more with Wash Buffer 2STEP 8. Dry the membranes of Filter Plate ．Gently tap the tips of the Filter Plate on a clean paper towel to remove residual liquid ．Apply vacuum for an addition 10 min.

STEP 9. RNA Elution ．Place a Elution Plate (provided, 96-Well PCR plate) on top of a clean 96-Well, 2 ml Plate (provided, the fourth 96-well, 2 ml plate) and place the Filter Plate on the Elution plate. (top: Filter Plate, middle: 96-well PCR Plate, bottom: 96-Well 2 ml plate) ．Add 50 ~ 75 μl of RNase-free Water to the membrane center of the Filter Plate. Stand for 3 min. ．Place the combined plate in a rotor bucket and centrifuge for 5 min at 5,600 – 6,000 x g for 5 min to elute RNA. ．Seal the Adhesive Film and store the RNA at -70 °C.

•(Optional STEP : Digest DNA by DNase I) Follow the steps from B1 ~ B4 to eliminate DNA. Otherwise, proceed to STEP 5 directly. •B1. Add 200 µl of Wash Buffer 1 (ethanol added) to each well of the Filter Plate. Apply vacuum at 10 inches Hg for 2 min. Discard the flow-through and return the Filter Plate to the manifold. •B2. Add 50 µl of RNase-free DNase I solution (0.5U/ul, not provided) to each well’s membrane of the Filter Plate. Stand the plate for 15 min at room temperature. •B3. Add 200 µl of Wash Buffer 1 to the to each well of the Filter Plate. Apply vacuum at 10 inches Hg until the wells have emptied. Discard the flow-through and return the Filter Plate to the manifold. •B4. After DNase I treatment, proceed to STEP 6.