MaestroZolTM RNA PLUS Extraction Reagent
Cat. No. MR-ZOL200
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Size: MaestroZol
Description:
MaestroZol
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x200 ml and PLUS reagent x 50mlStore at 4oC
RNA PLUS Extraction Reagent is a ready-to-use reagent for the isolation of total RNA from
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cells and tissues. The sample is homogenized or lysed in MaestroZol RNA plus extraction reagent and
then separated into organic, inter and aqueous phase, RNA can be precipitated by isopropyl alcohol from
aqueous phase.
The isolated RNA is suitable for any downstream application such us RT-PCR, in vivo translation and
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hybridization. MaestroZol RNA plus extraction reagent also included PLUS solution for polysaccharides
and proteoglycans elimination.
Reagents required, but not supplied
Chloroform
Isopropyl alcohol
75% Ethanol (in DEPC-treated water)
DEPC-treated Water
I Homogenization
Tissues:
Homogenize tissues samples (10-100mg) in 1ml of MaestroZol
Cells Grown in Monolayer:
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RNA plus extraction reagent.
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Lyse cells directly in culture dish or flask by adding 1 ml of MaestroZol RNA plus extraction reagent per 10
cm2dish. Pipette the cell lysate several times to ensure sufficient cell disruption.
* Too much samples or incomplete homogenization will cause contamination of genomic DNA and RNA
degradation after isolation.
* For some samples such as muscles tissue and plant tuberous which contain high amount of protein, fat,
polysaccharides or extracellular materials may not be dissolved in homogenate. Centrifuge the insoluble
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materials by 12,000 x g for 10 minutes at 4 C.
Cells Grown in Suspension:
Pellet cells at 200 x g for 5 minutes at room temperature, adding 1 ml of MaestroZol
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RNA plus extraction
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reagent per 5x10 –1x10 of cells. Pipette the cell lysate several times until complete lyses.
II RNA Isolation
Phase Separation:
1. Incubated the homogenate for 5 minutes at room temperature to completely dissociate the nucleoprotein
complex.
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2. Add 0.2 ml of chloroform per 1 ml of MaestroZol RNA plus extraction reagent. Cap and shake vigorously
for 15 seconds, then incubate at room temperature for 2-3 minutes.
3. Centrifuge the samples at 12,000 x g for 15 minutes at 4oC. After centrifugation, the sample will separates
into a yellow, phenol chloroform phase, an interphase and a colorless upper aqueous phase. RNA remains
exclusively in the aqueous phase.
RNA Precipitation:
RNA Precipitation for Polysaccharide-free Samples:
1. Transfer the aqueous phase into a new tube.
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2. Add 0.5ml of isopropyl alcohol of per 1 ml MaestroZol RNA plus extraction reagent and mix gently,
incubate at room temperature for 10 minutes then centrifuge samples at 12,000 x g for 10 minutes at 4oC.
(RNA precipitate forms a white pellet at the bottom of the tube).
RNA Precipitation for Polysaccharide-rich Samples:
1. Transfer the aqueous phase into a new tube.
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2. Add 0.25ml of isopropyl alcoholand0.25 ml PLUS solution of per 1 ml MaestroZol RNA plus extraction
reagent and mix gently, incubate at room temperature for 10 minutes then centrifuge samplesat 12,000 x g
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for 10 minutes at 4 C. (RNA precipitate forms a white pellet at the bottom of the tube).
* Note: PLUSSolution can effectivelyfacilitatepolysaccharide orproteoglycanremoval in the RNAprecipitation,
yetlow molecular weight RNAsuch as 5Smayalso be partial removed by PLUS Solutionsimultaneously.
RNA Wash:
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1. Remove the supernatant, wash the RNA pellet once with 1 ml of 75% ethanol per 1 ml MaestroZol
plus extraction reagent.
RNA
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2. Vortex and centrifuge at 7,500 x g for 5 minutes at 4 C, then remove the ethanol and air dry the RNA pellet
for 5-10 minutes.
Redissolving the RNA:
1. Dissolve RNA pellet in DEPC-treated Water by pipetting the solution up and down, and incubate for 10
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minutes at 55-60 C.
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2. Store RNA at -70 C.
III DNA Isolation
Reagents required, but not supplied:
100% Ethanol
0.1 M or HEPES (free acid ), no pH adjustment
0.1 M EDTA (pH8.0)
8 mM NaOH
0.1M sodium citrate in 10% ethanol
75% Ethanol
After complete removal of the aqueous phase for RNA isolation steps, carefully keep interphase and
organic phase for the use of following DNA isolation protocol. (Note: Completely removal of the aqueous
phase can reduce RNA contamination to minimum.)
For DNA precipitation, add 0.3 ml of 100% ethanol per 1 ml MaestroZolTM RNA plus extraction
reagentto the interphase and organic phase, and mix well by inverting several times.
Incubate the mixture at room temperature for 2-3 minutes, After that, centrifuge samplesat<2,000 x g
for 5 minutes at 4℃. (Note: Too high speed of centrifugation may cause DNA shearing.)A white DNA pellet
will be precipitated at the bottom of the tube after centrifugation. Discard the supernatant. Please carefully
remove aqueous phase to ensure the quality of isolated DNA.
If protein isolation is desired, transfer the supernatant to a new tube and store at 4℃, proceed to
Protein Isolation section at step 5.
Wash the DNA pellet by adding 1ml 0.1M sodium citrate in 10% ethanol per 1 ml MaestroZolTM RNA
plus extraction reagent. Incubate the DNA pellet in solution at room temperature for 30 minutes with
occasionallymixing during incubation to remove the phenol from pellet, andcentrifuge at <2,000g for 5
minutesat 4℃.
Repeat step 5 for additional one or two times wash in 1ml 0.1M sodium citrate-10% ethanol solution
based on the amount of DNA pellet.
After two or three washes, suspend the pellet in 1.5 ml of 75% ethanol per 1 ml MaestroZolTM RNA
plus extraction reagent and incubate at room temperature for 10 minutes and vortex 2-3 times during
incubation, then centrifuge at <2,000g for 5 minutes at 4℃.
Remove the supernatant, air dry the pellet for 5-10 minutes. Add 300-600μl of 8 mM NaOH to dissolve
the DNA pellet( DNA will not be dissolved in water or TE buffer ), incubate at 60℃and flicking the tube from
time to timeto facilitate DNA to dissolve.
* Do not dry the DNA pellet by SpeedVac, which will make DNA difficult to dissolve.
* DNAin 8 mM NaOH can only be stored overnight at 4℃.
*DNA may contain insoluble materials, remove themby centrifugation at 12,000 x g for 10 minutes and
transfer the supernatant to a new tube.
Adjust the pH of DNA solution to pH 7.5-8.0 by adding 12μl of 0.1 M HEPESand 1μl of 0.1 M EDTAper
100μl of DNA solution. Now the DNA can be stored at -20℃for months and is ready for PCR or restriction
digestions.
IVProtein Isolation
Reagents required, but not supplied:
Isopropyl alcohol
0.3M Guanidine hydrochloride in 95% ethanol
Ethanol
1% SDS
1. After complete removal of the aqueous phase for RNA isolation steps, carefully keep interphase and
organic phase for the use of following protein isolation protocol. (Note: Completely removal of the aqueous
phase can reduce RNA contamination to minimum.)
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2. Add 0.3 ml of 100% ethanolper 1 ml MaestroZol RNA plus extraction reagentto the interphase and
organic phases. Mix well by inverting several times.
3. Incubate the mixture for 2-3 minutes at room temperature, then centrifuge at <2,000 x g for 5 minutes at 4
℃.
4. Transfer the supernatant to a new tube and store at 4℃.
5. Precipitate proteins from the phenol-ethanol supernatant by adding 1.5 ml of isopropanol per 1 ml
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MaestroZol RNA plus extraction reagent(if using 1.5 ml microcentrifuge tube, aliquot the phenol-ethanol
supernatant into two 1.5 ml tubes and add 0.75 ml of isopropyl alcohol to each tubes ).
6. Incubate the mixture for 10 minutes at room temperature followed by centrifugation at 12,000 x g for 10
minutes at 4℃.
7. Remove the supernatant and wash the pellet by 2 ml of 0.3M guanidine hydrochloride in 95% ethanol per
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1 ml MaestroZol RNA plus extraction reagent( if using 1.5 ml microcentrifuge tube, add 1ml 0.3M
guanidine hydrochloride in 95% ethanol to each tube ).
8. Incubate the solution at room temperature for 20 minutes. During incubation, vortex several times to
remove the phenol from pellet followed by centrifugation at 7,500 x g for 5 minutes at 4℃.
9. Wash twice as above, and discard the supernatant.
10. Add 2 ml of ethanolto the pellet (if using 1.5 ml microcentrifuge tube, add 1ml of ethanol to each tube )
and incubate at room temperature for 20 minutes. After incubation, centrifuge at 7,500 x g for 5 minutes at 4
℃.
11. Vacuum dry the pellet for 5 to 10 minutes and dissolve the protein pellet in 100-200 μlof1% SDSat 50℃.
(Note: Solution may contain insoluble material, remove it by centrifugation at 12,000 x g for 10 minutes at 4
℃and transfer the supernatant to a new tube.)Store the protein sample at -20℃and is ready for use in
PAGE/western blotting