链霉亲和素使用说明

MagneSphere Magnetic Separation Products

1. Description ..........................................................................................................1

2. Product Components and Storage Conditions............................................2

3. General Protocol for mRNA Purification from Total RNA......................3

A.

B.

C.

D.

E.

F. Preparation of SA-PMPs for Use........................................................................4Annealing of Probe..............................................................................................5Preparation of Stock Solution ............................................................................5Washing of SA-PMPs...........................................................................................5Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids...................5Elution of mRNA..................................................................................................6

4. General Protocol for Capture of Biotinylated IgG.....................................6

5. Troubleshooting mRNA Isolation.................................................................7

6. Appendix .............................................................................................................8

A.

B.

C.

D. Characteristics of SA-PMPs................................................................................8Creating a Ribonuclease-Free Environment.....................................................8Composition of Buffers and Solutions..............................................................9Related Products.................................................................................................10

1. Description

Historically, bioseparations have been performed by extraction, precipitation,centrifugation, gel electrophoresis or column chromatography. The StreptavidinMagneSphere Paramagnetic Particles (SA-PMPs) and Magnetic SeparationStands offer significant advantages over these approaches. Separations arerapid, and multiple washes or rounds of purification can be completed quickly.These products are appropriate for many magnetics-based separation protocolsand are used most extensively as tools for mRNA purification.The Streptavidin MagneSphereParamagnetic Particles consist of a magnetitecore coated with streptavidin. The affinity of biotin for streptavidin (Kd = 10–15) is one of the strongest and most stable interactions in biology. Thus, theseparticles combine convenient magnetic separation technology with theversatility and high affinity of the biotin-streptavidin interaction. The particlescan be used with a wide variety of commercially available biotinylated reagentsincluding oligonucleotides, peptides, lectins, antibodies and enzymes. The Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

·

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1. Description (continued)

biotin-streptavidin interaction cannot be reversed under nondenaturing

conditions; therefore, we do not recommend the use of SA-PMPs for

applications in which the biotinylated molecules need to be recovered

from the SA-PMPs.

The MagneSphereStands and Streptavidin MagneSphereParamagnetic

Particles are used in the PolyATtractSystems to isolate mRNA directly fromcells or tissue (PolyATtractSystem 1000) or from total RNA (PolyATtract

Systems I–IV). These systems provide all of the reagents required for efficientcapture of mRNA (see Section 6.D).

The MagneSphereMagnetic Separation Stands can be used in conjunction withthe SA-PMPs and any of the PolyATtractSystems. The MagneSphereStands use the same samarium/cobalt magnet used in the PolyATtractSystems.

2. Product Components and Storage Conditions

Size

(15 × 0.6ml) 9ml

(1 × 25ml) 25ml

Size

1 each

Size

0.5ml

1.5ml

12 × 75mm

0.5ml

1.5ml

12 × 75mmCat.#Z5481Z5482Cat.#Z5410Cat.#Z5331Z5332Z5333Z5341Z5342Z5343Product Streptavidin MagneSphereParamagnetic ParticlesFor Laboratory Use.Product PolyATtract System 1000 Magnetic Separation StandProduct MagneSphere Technology Magnetic Separation Stand (two-position)MagneSphere Technology Magnetic Separation Stand (twelve-position)For use with both 15ml and 50ml tubes (includes an adapter for use with 15ml tubes).

Storage Conditions:Store the Magnetic Separation Stands and Adapter at

room temperature. Store the Streptavidin MagneSphereParamagnetic Particlesat 4°C. Do not freeze the Streptavidin MagneSphereParamagnetic Particles,as this will reduce their performance.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·www.promega.com Printed in USA.Revised 6/09

3. General Protocol for mRNA Purification from Total RNA

A schematic diagram of mRNA isolation from total RNA samples using theStreptavidin MagneSphereParamagnetic Particles is provided in Figure 1. Theprotocol described in this section is designed for use with up to 1mg of totalRNA, which requires 0.6ml of SA-PMPs. The procedure may be performed

conveniently using the 9ml size of the SA-PMPs (Cat.# Z5481), which is

provided as 15 tubes of 0.6ml each, and the MagneSphereMagnetic SeparationStand designed for 1.5ml microcentrifuge tubes (Cat.# Z5332). The appropriatemagnetic stand for other applications can be determined by the amount of

starting material (see Table 1). For other amounts of RNA, proportionally scalethe amounts of SA-PMPs and Biotinylated Oligo(dT) Probe.

Figure 1. Schematic diagram of mRNA isolation using the SA-PMPs.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·Fax 608-277-2516 ·www.promega.com Part# TB246Page 3

3. General Protocol for mRNA Purification from Total RNA (continued)Table 1. Selection of MagneSphereMagnetic Separation Stand.

Capture

Sample SizeVolume Range

5–35mg tissue180μl–1.26ml

35–100mg tissue1.26–3.6ml

100mg–1g tissue3.6–36ml

551 × 10–2.5 × 10cells 110–275μl

562.5 × 10–1 × 10cells 275–1,100μl

1 × 106–1 × 108cells 1.1–18mlStand Tube Size(2-position)1.5ml Z533212 × 75mmZ533315ml/50ml*Z54100.5ml Z53311.5ml Z533215ml/50ml*Z5410Stand (12-position)Z5342Z5343NA Z5341Z5342NA

caps (e.g., Corningtubes) for sample sizes ³3.6ml. NA: not applicable.

Materials to Be Supplied by the User

(Solution compositions are provided in Section 6.C.)

•nuclease-free SSC: 20X, 0.5X and 0.1X

•water bath or heat block at 65°C

•autoclaved, RNase-free plastic tubes, 1.5ml

•sterile, RNase-free pipettes and pipette tips

•Biotinylated Oligo(dT) Probe, 50pmol/μl (Cat.# Z5261)

•Nuclease-Free Water (Cat.# P1193)

3.A. Preparation of SA-PMPs for Use

The SA-PMPs require BSA for stabilization, which is present in the storage

buffer and is removed once the particles are washed. The SA-PMPs are

provided at a concentration of 1mg/ml in a solution of PBS, 1mg/ml BSA and

0.02% sodium azide. The particles should be rinsed three times each with an

equal volume of 0.5X SSC and used within 30 minutesafter washing to

maintain optimal performance. The particles cannot be washed and reused

after the initial use.

The SA-PMPs must be completely resuspended to ensure adequate

performance. Discard particles that appear to have “clumped” and cannot be

dispersed. To determine if the particles are in good condition, mix by inverting

the tube several times and verify that the particles remain in suspension for at

least 3 minutes in a 0.5–1ml volume. If some of the particles settle out of

suspension within 3 minutes, forming an easily visible pellet, they should not

be used. To prevent clumping of the particles, do not freeze them or allow

them to dry out.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·www.promega.com Printed in USA.Revised 6/09

3.B. Annealing of Probe

1.

In an autoclaved, RNase-free tube, bring 0.1–1.0mg of total RNA to a finalvolume of 500μl in Nuclease-Free Water.

Note:Less total RNA (50μg) may be used, but the mRNA obtained may notbe detectable by spectrophotometry. Specific messages in small quantitiesof mRNA may be detected by RT-PCR.

2. Place the tube in a heat block at 65°C for 10 minutes.

3. Add 3μl of Biotinylated Oligo(dT) Probe and 13μl of 20X SSC to the RNA.Mix gently, and incubate at room temperature until completely cooled.This will require 10 minutes or less. While this solution is cooling, preparestock solutions of 0.5X and 0.1X SSC.3.C. Preparation of Stock Solution1. Prepare 1.2ml of nuclease-free 0.5X SSC by combining 30μl of 20X SSC with1.170ml of Nuclease-Free Water in a fresh, RNase-free tube.

2. Prepare 1.4ml of nuclease-free 0.1X SSC by combining 7μl of 20X SSC with1.393ml of Nuclease-Free Water in a fresh, RNase-free tube.

3.D. Washing of SA-PMPs

1. Resuspend each 0.6ml tube of SA-PMPs by gently flicking the bottom ofthe tube until the particles are completely dispersed, and capture them byplacing the tube in the magnetic stand until the SA-PMPs have collected atthe side of the tube (approximately 30 seconds).

2. Carefully remove the supernatant. Do not centrifuge the particles.

3. Wash the SA-PMPs three times with 0.5X SSC (300μl per wash). After eachwash, capture the SA-PMPs using the magnetic stand and carefully removethe wash solution.

4. Resuspend the washed SA-PMPs in 100μl of 0.5X SSC.

3.E. Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids

1. Add the entire contents of the annealing reaction (Section 3.B, Step 3) to thetube containing the washed SA-PMPs.

2. Incubate at room temperature for 10 minutes. Gently mix by inversionevery 1–2 minutes.

3. Capture the SA-PMPs using the magnetic stand, and carefully remove thesupernatant without disturbing the SA-PMPs.

Save the supernatant from Step 3 until you are certain that satisfactorybinding and elution of mRNA has occurred.

4. Wash particles four times with 0.1X SSC (300μl per wash) by gently flickingthe bottom of the tube until all particles are resuspended, then capturingthe particles using the magnetic stand. After the final wash, remove asmuch liquid as possible without disturbing the SA-PMPs.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·Fax 608-277-2516 ·www.promega.com Part# TB246Page 5

3.F. Elution of mRNA

1. To elute the mRNA, add 100μl of Nuclease-Free Water to the SA-PMPs and

gently resuspend the particles by flicking the tube.

2. Magnetically capture the SA-PMPs, and transfer the eluted mRNA to a

fresh RNase-free tube. Do not discard the particles.

Note:If any particles were transferred, remove by centrifugation at

12,000×g for 5–10 minutes at 4°C. Carefully transfer the RNA to a fresh

RNase-free tube.

3. Repeat the elution by resuspending the SA-PMPs in 150μl of Nuclease-Free

Water. Repeat the capture step, pooling the eluate with RNA eluted in

Step 2 (250μl total volume). Save the SA-PMPs in 100μl of Nuclease-Free

Water until you have verified that the mRNA has been eluted satisfactorily.

4. General Protocol for Capture of Biotinylated IgG

The following is a typical protocol to capture a biotinylated antibody using theSA-PMPs. The protocol uses biotinylated rabbit IgG (e.g., Pierce Cat.# 31826)

but can be used with other biotinylated antibodies as well. The reaction

requires 1mg of antibody and 10ml of SA-PMPs (at 1mg/ml), but thesequantities may be scaled proportionally depending on the amount of antibodyused. As a general guideline, 1mg of SA-PMPs will bind at least 70μg of

biotinylated rabbit IgG.

Materials to Be Supplied by the User

(Solution compositions are provided in Section 6.C.)

•PBS, 1X

•biotinylated antibody preparation

1. Prepare the biotinylated IgG in 1X PBS. If necessary, remove any insoluble

material by centrifugation at 12,000 × g in a microcentrifuge for a few seconds at

room temperature. An antibody concentration of 4mg/ml is used in this protocolbut can be varied depending on the application.

2. Resuspend SA-PMPs (1mg/ml) by gently flicking and inverting the bottle untilthe particles are completely dispersed. Dispense 10ml of SA-PMPs into an

appropriately sized tube. Place the tube in the magnetic stand until the SA-PMPshave collected at the side of the tube (approximately 30 seconds). Carefully

remove the supernatant. Do not centrifuge the particles.

3. Wash SA-PMPs three times, each with 10ml of 1X PBS (use a volume equal to theinitial volume of SA-PMPs for each wash), capturing the particles using the

magnetic stand, then carefully removing the supernatant each time .

4. Resuspend the washed SA-PMPs in 1ml of 1X PBS. The final concentration of

particles is now 10mg/ml.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·www.promega.com Printed in USA.Revised 6/09

5. Add 1mg (250μl) of antibody preparation from Step 1 to 1ml of concentrated

SA-PMPs (from Step 4). Incubate at room temperature for 30 minutes with gentlerotation of the tube.

6. After the 30-minute binding reaction is complete, magnetically capture the

SA-PMP-antibody complexes, carefully remove the supernatant and place in a

clean tube.

Note:The supernatant can be assayed for unbound IgG to determine the amountof IgG bound to the SA-PMPs. Centrifuge the supernatant at 12,000 × g for

5minutes to pellet any SA-PMPs that may have been transferred. The protein

concentration in the supernatant can be determined spectrophotometrically by

using a protein assay or by immunoassay.

7. Wash the SA-PMP-antibody complexes three times, each with 10ml of 1X PBS.After each wash, capture particles using the magnetic stand and carefully

remove the wash solution.

8. Resuspend the SA-PMP-antibody complexes in the appropriate buffer for yourapplication.

5. Troubleshooting mRNA Isolation

For questions not addressed here, please contact your local Promega Branch Office or Distributor.Contact information available at: www.promega.com . E-mail: [email protected]

Problem

No mRNA elutedCauses and CommentsNo mRNA bound because salt was omitted from annealing procedure. Repeat the annealing step,

adding 20X SSC to a final concentration of 0.5X.

Insufficient cooling of annealing reaction before

probe capture and wash. Add the saved supernatant back to the particles as in Section

3.E, Step 1, and continue with the procedure.

Salt was not eliminated before elution. Wash theSA-PMPs again with Nuclease-Free Water, and

check the A260of this eluate.

RNase contamination in total RNA. Evaluate the quality of total RNA by gel electrophoresis,

and repeat the total RNA isolation as necessary.

RNA appears degraded on gelRNase contamination during mRNA isolation. Repeat the entire procedure using a fresh total

RNA sample.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

·Fax 608-277-2516 ·www.promega.com Part# TB246Page 7

6. Appendix

The SA-PMPs are approximately 1.0μm in diameter and are irregularly

shaped, resulting in a surface area (100–150m2/g) that is 20–30 times greater

than that of spherical particles with a similar diameter. One milligram of

SA-PMPs will bind approximately 1nmol of biotinylated oligonucleotide or

100

μ

g of biotinylated IgG.

Promega SA-PMPs are quality tested with both antibodies and nucleic acids.

The SA-PMPs are free of DNase and RNase activity as determined by assays

using [3H]DNA and [3H]RNA templates. The binding capacity is measured

using biotinylated oligo(dT) and biotinylated rabbit IgG. The SA-PMPs also

are tested for mRNA isolation from mouse liver using the PolyATtract

System 1000.

•

•

•

•

•Size:The SA-PMPs have an average diameter of 1.0 ± 0.5μm. pH Stability:The SA-PMPs are stable over a pH range of 5.0–9.0.Temperature Stability:The particles are stable over a temperature range

of 4–65°C. Temperatures outside this range will cause the particles to

clump and to be destroyed.

Effect of SDS:A decrease in performance of the SA-PMPs is observed at

SDS concentrations >5.0%.6.A. Characteristics of SA-PMPs•

6.B. Creating a Ribonuclease-Free Environment

Ribonucleases are extremely difficult to inactivate. Great care should be taken

to avoid inadvertently introducing RNases into an RNA preparation during or

after isolation. This is especially important if the starting material was difficult

to obtain or is irreplaceable. The following notes may be helpful in preventing

the accidental contamination of your sample.

1. Two of the most common sources of RNase contamination are the user’s

hands and bacteria or molds that may be present on airborne dust particles

or laboratory glassware. To prevent contamination from these sources,

sterile technique should be used when handling any reagents used for

RNA isolation or analysis. Gloves should be worn at all times.

2. Whenever possible, sterile disposable plasticware should be used when

handling RNA samples. These materials are generally RNase-free and do

not require pretreatment to inactivate RNases.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·www.promega.com Printed in USA.Revised 6/09

3.

Nondisposable glassware and plasticware should be treated before use to

ensure that it is RNase-free. Glassware should be baked at 200°C overnight,

and plasticware should be thoroughly rinsed before use with 0.1N NaOH/

1mM EDTA, then with diethyl pyrocarbonate (DEPC)-treated water.

4. Autoclaving alone is not sufficient to inactivate RNases. COREXtubes

should be rendered RNase-free by treatment with DEPC and not by

baking. This will reduce the failure rate of this type of tube during

centrifugation. Solutions and COREXtubes supplied by the user should

be treated with 0.1% DEPC overnight at room temperature, then

autoclaved for 30 minutes to remove any trace of DEPC.

Tris buffers cannot be treated with DEPC. Use caution when weighing Tris

to avoid RNase contamination, and use DEPC-treated water and glassware

when preparing Tris buffers.

5. While most sources of fresh deionized water are free of contaminating

RNase activity, deionized water sources can be a potential contributor of

RNase activity. If degradation of the target RNA occurs, it may be

necessary to test the laboratory water source for RNase activity.

6. We recommend that chemicals used for RNA isolation and analysis be

reserved solely for this purpose. Wear gloves when handling these

chemicals, and use only baked spatulas and untouched weigh boats or

weigh paper.

6.C. Composition of Buffers and Solutions

DEPC-treated water

Add diethyl pyrocarbonate (DEPC)

to deionized water to a final

concentration of 0.1% (v/v).

Incubate overnight at room

temperature in a fume hood.

Autoclave for 20 minutes.PBS, 1X0.2g 8.0g 0.2g 1.15g KCl NaCl KH 2PO 4Na 2HPO 4

DEPC is a suspected carcinogen.

Work in a fume hood, and follow

standard laboratory safety

procedures.

SSC, 20X

87.7g NaCl

44.1g trisodium citrate

dihydrate

Dissolve in 400ml of DEPC-treated

water. Adjust the pH to 7.2 with

10N HCl, and bring the final

volume to 500ml. Dispense into

aliquots. Sterilize by autoclaving.Add components one at a time to900ml of room-temperaturedeionized water, and mix untilcompletely dissolved. Adjust the pHto 7.4 using 1N HCl or 1N NaOH, ifnecessary. Bring the final volume to1 liter. If stored for long periods,filter the solution through a 0.45μm filter and store in a tightly cappedbottle.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA ·Fax 608-277-2516 ·www.promega.com Part# TB246Page 9

6.D. Related Products

Systems for mRNA Purification from Total RNA

Product

PolyATtract mRNA Isolation System I (Refill System for Z5200)PolyATtract mRNA Isolation System II

Cat.#Z5210Z5200

For Laboratory Use. Each system contains sufficient reagents for three separate mRNAisolations, each from 1–5mg total RNA. Note:Cat.# Z5210 does notcontain the magneticseparation stand.

Product

PolyATtract mRNA Isolation System III

PolyATtract mRNA Isolation System IV (Refill System for Z5300)Cat.#Z5300Z5310

For Laboratory Use. Each system contains sufficient reagents for fifteen separate mRNAisolations, each from 100–1,000μg total RNA. Note:Cat.# Z5310 does notcontain themagnetic separation stand.

Systems for mRNA Isolation from Biological SamplesProduct

PolyATtract System 1000

PolyATtract System 1000 (Refill System for Z5420)

Cat.#Z5420Z5400

For Laboratory Use. Each system contains sufficient reagents for mRNA isolation from up to2g of tissue or 4 × 108cultured cells. Note:Cat.# Z5400 does notcontain the magneticseparation stand.

Systems for Total RNA IsolationProduct

PureYield RNA Midiprep SystemSV Total RNA Isolation SystemFor Laboratory Use.

Size 10 preps50 preps50 prepsCat.#Z3740Z3741Z3100

Amplification-Related ProductsProduct

Access RT-PCR SystemFor Laboratory Use.

Size

100 reactions500 reactionsSize

100 reactions1,000 reactions

Cat.#A1250A1280Cat.#M7122M7123

Product

GoTaq Green Master Mix

For Laboratory Use. GoTaqGreen Master Mix is a premixed solution of GoTaqDNA Polymerase, GoTaqGreen Reaction Buffer, dNTPs and Mg2+. Catalog numbers may be

different in Europe.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

·www.promega.com

Printed in USA.Revised 6/09

Amplification-Related Products (continued)Product

GoTaq DNA Polymerase

Conc. 5u/μl

Size 100u

Cat.#M3001

For Laboratory Use. GoTaqDNA Polymerase contains 5X Green GoTaqReaction Bufferand 5X Colorless GoTaqReaction Buffer. Both buffers provide 1.5mM MgCl2in the final1X concentration. Available in additional sizes. Catalog numbers may be different inEurope.

Product

GoTaq Flexi DNA PolymeraseConc. 5u/μl Size 100u Cat.#M8291

For Laboratory Use. GoTaqFlexi DNAPolymerase contains 5X Green GoTaqFlexi Buffer, 5X Colorless GoTaqFlexi Buffer and Magnesium Chloride Solution, 25mM.

Reaction buffers are magnesium-free. Available in additional sizes. Catalog numbers maybe different in Europe.

Product

ImProm-II Reverse Transcription System*ImProm-II Reverse Transcriptase*

M-MLV Reverse Transcriptase*

M-MLV Reverse Transcriptase, RNase H Minus**M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant**

AMV Reverse Transcriptase*

Recombinant RNasinRibonuclease Inhibitor**For Laboratory Use.

**Not available for purchase in the United States.

Size

100 reactions10 reactions100 reactions500 reactions

10,000u 10,000u 10,000u 300 units2,500 units

Cat.#A3800A3801A3802A3803M1701M5301M3682M5101N2511

Other Related Products

Product

MagnaBot 96 Magnetic Separation DeviceBiotinylated Oligo(dT) Probe (50pmol/μl) RNA Markers, 0.28–6.58kbFor Laboratory Use.

Size

96-well plate

35μl 50μl Cat.#V8151Z5261G3191

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

·Fax 608-277-2516 ·www.promega.com

Part# TB246

Page 11

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GoTaq, MagnaBot, MagneSphere, PolyATtract, RNAgents and RNasin are registered trademarks of Promega Corporation.ImProm-II and PureYield are trademarks of Promega Corporation.COREX and Corning are registered trademarks of Corning, Inc.

Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for moreinformation.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the

most up-to-date information on Promega products.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

·www.promega.com

Printed in USA.Revised 6/09