favorprep 96-well total rna kit - · pdf filefavorprep 96-well total rna kit - for isolation...
TRANSCRIPT
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.