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psicheck2质粒说明书

psicheck2质粒说明书
psicheck2质粒说明书

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1.Description ..........................................................................................................12.Product Components and Storage Conditions ............................................23.General Considerations (3)

A.siCHECK? Vector Features...............................................................................3B.How the siCHECK? Vectors Work..................................................................4C.Sample Experiments Using the siCHECK? Vectors.. (6)

4.siCHECK? Vector Maps .................................................................................95.siCHECK? Vector Restriction Enzyme Tables . (11)

A.Restriction Enzyme Sites for the psiCHECK?-1 Vector..............................11B.Restriction Enzyme Sites for the psiCHECK?-2 Vector (13)

6.siCHECK? Vector Backbones and Components .....................................15

7.References .........................................................................................................16

8.Related Products ..............................................................................................181.

Description

The psiCHECK?-1 Vector (a–d)(Cat.# C8011) and psiCHECK?-2 Vector (a–f)(Cat.# C8021) are designed to provide a quantitative and rapid approach for optimization of RNA interference (RNAi). The vectors enable the monitoring of changes in expression of a target gene fused to the reporter gene. In both vectors, Renilla luciferase is used as a primary reporter gene, and the gene of interest can be cloned into the multiple cloning region located downstream of the Renilla luciferase translational stop codon. Initiation of the RNAi process toward a gene of interest results in cleavage and subsequent degradation of fusion mRNA. Measurement of decreased Renilla luciferase activity is a convenient indicator of RNAi effect (1).

RNAi is a phenomenon by which double-stranded RNA complementary to a target mRNA can specifically inactivate gene function by stimulating the degradation of the target mRNA (2–4). Because of the ability to inactivate genes, RNAi has emerged as a powerful tool for analyzing gene function.

siCHECK? Vectors

All technical literature is available on the Internet at: https://www.sodocs.net/doc/522437431.html,/tbs Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use

of this system. E-mail: techserv@https://www.sodocs.net/doc/522437431.html,

In mammalian systems, including cultured mammalian cells, chemically

synthesized double-stranded short interfering RNA molecules (<30 nucleotides;

siRNA) or endogenously expressed short hairpin RNA molecules (shRNA) result in dsRNA duplexes <30 base pairs in length that induce RNAi (5–10). RNAi

duplexes >30bp induce the interferon response and nonspecific degradation of

mRNA and cannot be used as tools for specific gene silencing (11,12).

Interestingly, a significant percentage of the siRNA or shRNA designed for a

specific gene are not effective (5,13–16). On average only 1 in 5 of the

siRNA/shRNAs selected for targeting a specific region show efficient gene

silencing (16,17). Possible causes for the failure of a particular siRNA/shRNA

may be instability of an siRNA probe in vivo, inability to interact with

components of the RNAi machinery or the inaccessibility of the target mRNA

due to local secondary structural constraints. Analysis of nucleotide sequences,

melting temperatures and secondary structures have not revealed any obvious

difference between effective and ineffective siRNA/shRNA (18).

At present, one of the most serious limitations for the RNAi technology is the

lack of a rapid, reliable, quantitative target-site screening method. Various

algorithm programs exist that aid in the design of potential siRNA targets.

However, an experimental method is needed to screen these siRNAs. Current

screening technologies include such semi-quantitative, time-consuming methods as fluorescence change for GFP-target fusions, Western blot analysis, monitoring

phenotypic changes or RT-PCR. In addition, the current screening technologies

are not easily modified for the rapid, simultaneous screening of multiple

siRNA/shRNA.

2.Product Components and Storage Conditions

Product Size Cat.# psiCHECK?-1 Vector20μg C8011 psiCHECK?-2 Vector20μg C8021 Storage Conditions:Store the psiCHECK?-1 and psiCHECK?-2 Vectors at

–20°C.

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3.General Considerations

3.A. siCHECK? Vector Features

Current methods to monitor changes in gene expression as the result of RNAi are either semi-quantitative, time-consuming or not applicable to high-throughput screening. The siCHECK? Vectors are easier to use than currently available methods, allow optimal quantitative target site selection and can be adapted for use in high-throughput methodologies.

There are two siCHECK? Vectors, the psiCHECK?-1 Vector and the

psiCHECK?-2 Vector. Both vectors contain as the primary reporter gene the synthetic version of Renilla luciferase, hRluc , which is used to monitor changes in expression as the result of RNAi induction. This synthetic gene is engineered for more efficient expression in mammalian cells and for reduced anomalous transcription.

To aid in fusion of the target gene to the synthetic Renilla luciferase reporter gene, a region of restriction sites (i.e., the multiple cloning region) has been added 3′ to the Renilla translational stop. The restriction sites present in the multiple cloning region can be used to create genetic fusions between the gene of interest and the Renilla reporter gene. Because no fusion protein is

expressed, there is no need to be concerned about whether you have cloned into a proper translational reading frame.

The multiple cloning region of the psiCHECK?-1 Vector contains unique restriction sites SgfI, XhoI, SmaI, EcoRI, PmeI and NotI. Due to the presence of the firefly expression cassette, the psiCHECK?-2 Vector contains fewer unique restriction sites. The restriction sites in the psiCHECK?-2 Vector multiple cloning region are SgfI, XhoI, PmeI and NotI.

The promoter used for Renilla luciferase expression in the siCHECK? Vectors is the SV40 promoter. Experimental results (data not shown) demonstrate that the SV40 promoter results in the best balance between Renilla luciferase

expression and the detection of RNAi activity when used with siRNA or vectors expressing shRNA.

The difference between the two siCHECK? Vectors is that the psiCHECK?-2Vector possesses a secondary firefly reporter expression cassette. The firefly expression cassette consists of an HSV-TK promoter, a synthetic firefly luciferase gene and an SV40 late poly(A) signal. To reduce the potential for recombination events, the Renilla luciferase reporter gene in the psiCHECK?-2Vector uses a synthetic poly(A). This firefly reporter cassette has been

specifically designed to be an intraplasmid transfection normalization reporter;thus when using the psiCHECK?-2 Vector, the Renilla luciferase signal can be normalized to the firefly luciferase signal.

If no transfection normalization is required or one would prefer to have the transfection normalization reporter on a second plasmid, the psiCHECK?-1Vector is the vector of choice.

3.A. siCHECK? Vector Features (continued)

The psiCHECK?-1 Vector is recommended for use in monitoring RNAi

effects in live cells. The changes in Renilla luciferase activity are measured

with EnduRen? Live Cell Substrate (Cat.# E6481), which allows continuous

monitoring of intracellular Renilla luminescence (19; Figure 2). EnduRen? Live

Cell Substrate is for use only with Renilla luciferase.

Promega offers several reagents that can be used in conjunction with the

siCHECK? Vectors to monitor Renilla and/or firefly luciferase signals. For the

psiCHECK?-1 Vector, which only contains the Renilla luciferase reporter gene,

the Renilla Luciferase Assay System (Cat.# E2810, E2820) can be used. The

psiCHECK?-2 Vector, which contains Renilla and firefly luciferase reporter

genes, requires the use of either the Dual-Luciferase?Reporter Assay System

(Cat.# E1910) or the Dual-Glo? Luciferase Assay System (Cat.# E2920) to

generate the firefly and Renilla luciferase signals.

3.B. How the siCHECK? Vectors Work

Figure 1 provides a basic description of how the siCHECK? Vectors work.

Using the unique restriction sites, the gene of interest is cloned into the

multiple cloning region located 3′ to the synthetic Renilla luciferase gene and

its translational stop codon. After cloning, the vector is transfected into the

mammalian cell line of choice, and a fusion of the Renilla luciferase gene and

the gene of interest is transcribed. Vectors expressing potential shRNA or

siRNA can be cotransfected simultaneously or sequentially, depending on

your experimental design. If a specific shRNA/siRNA binds to the target

mRNA and initiates the RNAi process, the fused Renilla luciferase:gene of

interest mRNA will be cleaved and subsequently degraded, decreasing the

Renilla luciferase signal.

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translation stop

cleavage of mRNA

light mRNA

5′3′5′

3′

4339M A 10_3A

5′3′hRluc

gene of interest

hRluc Figure 1. Mechanism of action of the siCHECK? Vectors.

3.C. Sample Experiments Using the siCHECK? Vectors

To demonstrate the utility of the siCHECK? Vectors, two experiments are detailed in this Technical Bulletin. In the first experiment, human p53 cDNA was subcloned into the psiCHECK?-1 and the psiCHECK?-2 Vectors using the SgfI and NotI restriction sites located in the multiple cloning region of both vectors. Note the SgfI and NotI restriction sites are located 3′ to the Renilla luciferase translational stop codon. As shown in Figure 2, the psiCHECK?-1Vector containing the human p53 cDNA was cotransfected into HEK-293T cells with the psiLentGene? Basic Vector expressing either a Renilla luciferase (hRluc ) or p53 shRNA. The negative control was the psiLentGene? Basic Vector with a nonspecific 19bp insert. (A BLAST search using this 19bp sequence and a threshold >90% revealed no homology to any known

mammalian gene or to the synthetic Renilla luciferase gene.) This nonspecific

sequence was used for all RNAi experiments in this Technical Bulletin.

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4398M A 11_3A

R e n i l l a L u m i n e s c e n c e (R L U )

Time Post-Transfection (hours)

Negative Control Renilla p53

Figure 2. Inhibition of Renilla luciferase expression by targeting either the Renilla luciferase or p53 gene.The human p53 cDNA was subcloned into the psiCHECK?-1Vector using the SgfI and NotI restriction sites located 3′ to the Renilla luciferase translational stop codon. To begin the transfection assay, HEK-293T cells were plated in a 96-well plate at 3,000 cells/well. After an overnight incubation, the cells were treated with a transfection mixture consisting of 35μl of serum-free medium,0.3μl of TransFast? Transfection Reagent (Cat.# E2431), 0.02μg of psiCHECK?-1:p53vector and 0.08μg of psiLentGene? Basic Vector per well. For this experiment, the psiLentGene? Vector expressed shRNAs directed against human p53, Renilla luciferase or the nonspecific 19bp sequence, which serves as a negative control,(Section 3.C). After a one-hour incubation, 100μl of serum-containing medium was added to the wells. At 21 hours post-transfection, EnduRen? Live Cell Substrate (Cat.# E6481) was added to a final concentration of 60μM, and Renilla luciferase activity was monitored. Renilla luciferase activities were normalized to the number of viable cells using the CellTiter-Glo ?Luminescent Cell Viability Assay (Cat.#G7573; 20).

At 21 hours post-transfection, nonlytic EnduRen? Live Cell Substrate was

added to the wells; luminescence was monitored for the next 27 hours until

48 hours post-transfection. The data in Figure 2 show that the psiLentGene?

Basic Vector expressing either Renilla luciferase or p53 shRNA inhibits the

expression of the Renilla luciferase reporter gene from the psiCHECK?-1:p53

vector. Interestingly, using either Renilla luciferase or p53 shRNA results in

virtually identical inhibition of Renilla luciferase expression.

In a second experiment, the human p53 cDNA used in Figure 2 was subcloned

into the psiCHECK?-2 Vector using the SgfI and NotI restriction sites. Five

potential p53 shRNAs designed to bind to five different target sites were cloned

into the psiLentGene? Basic Vector; the resulting vectors were named Site 1

through Site 5. The control is a psiLentGene? Vector containing the nonspecific

19bp sequence. The psiCHECK?-2 Vector containing the p53 cDNA was

cotransfected with the psiLentGene? Vector expressing either a p53 shRNA

(Figure 3, Sites 1–5) or the nonspecific shRNA into HEK-293T cells as described

in Figure 3. Forty-eight hours after transfection, the medium was removed and

cells were lysed in Passive Lysis Buffer (Cat.# E1941). The firefly and Renilla

luciferase signals were generated using the Dual-Luciferase?Reporter 1000

Assay System (21).

Figure 3, Panel A, displays the Renilla luciferase signal, while Figure 3, Panel B,

shows the Renilla luciferase signal normalized (corrected for transfection

efficiency to the firefly luciferase signal). The data in Figure 3, Panel A, is

difficult to interpret due to transfection variations. The Renilla luciferase

positive control, which should demonstrate inhibition of reporter expression, is

not statistically different (i.e., overlapping error bars) from the negative control

(no effect on reporter expression was detected). The inability to distinguish

between the positive and negative controls renders any conclusion regarding

the effectiveness of potential shRNAs suspect.

However, when the Renilla luciferase signals are normalized (see Figure 3,

Panel B) to the internal firefly luciferase transfection control, the data

interpretation is different, as the Renilla luciferase positive control is

statistically different from the negative control. In addition, the normalized

data allow the ability to distinguish the effectiveness of the various target site

shRNAs.

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Figure 3. Target site selection using the psiCHECK?-2 Vector. HEK-293T cells were seeded into a 96-well plate at a density of 3,000 cells/well. Human p53 cDNA was subcloned into the psiCHECK?-2 Vector using the SgfI and NotI restriction sites. After an overnight incubation, the cells were treated with a transfection mixture consisting of 35μl of serum-free medium, 0.3μl of TransFast? Transfection Reagent (Cat.# E2431), 0.02μg of psiCHECK?-2 Vector:p53 and 0.08μg of psiLentGene?Basic Vector per well. The psiLentGene? Basic Vector expressed one of five different shRNAs directed against human p53, Renilla luciferase or a nonspecific 19bp sequence (Section 3.C) as a negative control. After a one-hour incubation, 100μl of serum-containing medium was added to the wells. Forty-eight hours post-transfection Renilla and firefly luciferase activities were measured using the Dual-Luciferase ?Reporter 1000 Assay System (Cat.# E1980; 21). Panel A displays the raw Renilla luciferase data, while in Panel B , the Renilla luciferase data has been

normalized to firefly luciferase data. The data represent the mean of 12 wells plus or minus the standard deviation. Note that in other experiments the ability of different shRNAs to inhibit gene expression might vary more dramatically.

4399M A 11_3A

A.

B.S i t e

1

S i t e

2

S i t e

3

S i t e 4 S i t e

5 R e n

i l l a P o s i

t i v

e C o n t r o l N

e g a t i v e C o n t r o l R e n i l l a L u m i n e s c e n c e (R L U )

S i t e 1

S i t e 2

S i t e 3 S i t e 4 S i t e 5 R e n i l l a P

o s i t i v e C o n

t r o l N e g a t i v e C o n t r o l

N o r m a l i z e d R e n i l l a L u m i n e s c e n c e (R L U )

4.siCHECK? Vector Maps

psiCHECK?-1 Vector sequence reference points: SV40 early enhancer/promoter 7–425Chimeric intron

489–621T7 RNA polymerase promoter

666–684Synthetic Renilla luciferase gene (hRluc )694–1629Multiple cloning region 1636–1680Synthetic poly(A)

1688–1736β-lactamase (Amp r ) coding region

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4343M A 10_3A

BglII 1Figure 4. psiCHECK?-1 Vector map. –^– denotes the intron.Synthetic poly(A) signal

4342M A 10_3A

CCCGGGAATTCGTTTAAACCTAG

AGCGGCCGCTGGCCGC AATAAAATA . . . 3′

5′ . . . GAGCAGTAA TTCTAGGCGATCGCTCGAG XhoI

SmaI

NotI

EcoRI

PmeI

SgfI

hRluc

Figure 5. psiCHECK?-1 Vector multiple cloning region.

psiCHECK?-2 Vector sequence reference points:SV40 early enhancer/promoter 7–425Chimeric intron

489–621T7 RNA polymerase promoter

666–684Synthetic Renilla luciferase gene (hRluc )694–1629Multiple cloning region 1636–1680Synthetic poly(A)1688–1736HSV-TK promoter

1744–2496Synthetic firefly luciferase gene (hluc +)2532–4184SV40 late poly(A)

4219–4440β-lactamase (Amp r ) coding region

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Figure 6. psiCHECK?-2 Vector map.–^– denotes the intron.4345M A 10_3A

BglII 1Synthetic poly(A) signal

4344M A 10_3A

CCCGGGAATTCGTTTAAACCTAG

AGCGGCCGCTGGCCGC AATAAAATA . . . 3′

5′ . . . GAGCAGTAA TTCTAGGCGATCGCTCGAG

XhoI

NotI

PmeI

SgfI

hRluc

Figure 7. psiCHECK?-2 Vector multiple cloning region.

5.siCHECK? Vector Restriction Enzyme Tables

5.A.Restriction Enzyme Sites for the psiCHECK?-1 Vector

The following restriction enzyme tables were constructed using DNASTAR ?sequence analysis software. Please note that we have not verified this information by restriction digestion with each enzyme listed. The location given specifies the 3′-end of the cut DNA (the base to the left of the cut site).For more information on the cut sites of these enzymes, or to report a

discrepancy, please contact your local Promega Branch or Distributor. In the U.S., contact Promega Technical Services at 800-356-9526. Vector sequences are available from the GenBank ?database (GenBank ?/EMBL accession number AY535006) and online at:

https://www.sodocs.net/doc/522437431.html,/vectors/

Enzyme # of Sites Location AatII 11391Acc65I 154

AcyI 21388, 2121AflII 2452, 649Alw44I 21989, 3235AlwNI 13140

AspHI 41091, 1993, 2078,3239

AvaI 3715, 1643, 1649AvaII 22297, 2519AvrII 1404BamHI 11738

BanI 354, 575, 2708BanII 3759, 899, 1650BbsI 1560BbuI 2152, 224BclI 2734, 1187BglI 3357, 694, 2543BglII 11

BsaI 3514, 1234, 2595BsaOI

5

1640, 1677, 2143,2292, 3215BsaBI 11453

BsaHI 21388, 2121BspHI 21821, 2829BspMI 1476

BssSI 21992, 3376Bst98I 2452, 649BstZI 11280Cfr10I 1

2576

Enzyme # of Sites Location

DraI 41663, 2083, 2775,2794DraII 11539DraIII 1882

DrdI 2441, 3447

DsaI 415, 311, 692, 899EaeI 31674, 1681, 2268EagI 11674

EarI 21193, 1862EclHKI 12661Eco52I 11674Eco81I 11280EcoRI 11654EcoRV 11179FspI 28, 2438HaeII 13309

HgaI 41570, 2129, 2859,3437HindIII 1420

Hsp92I 21388, 2121KpnI 158

MspA1I 580, 1679, 2025,2966, 3211

NciI 51650, 1651, 2125, 2476, 3172NcoI 315, 311, 692NheI 1684NotI 11674NruI 11355

NsiI

3

154, 226, 913

Table 1. Restriction Enzymes That Cut the psiCHECK?-1 Vector Between 1 and 5 Times.

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5.A.

Restriction Enzyme Sites for the psiCHECK?-1 Vector (continued)Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

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Table 2. Restriction Enzymes That Do Not Cut the psiCHECK?-1 Vector.A ccB7I AccI AccIII AflIII AgeI ApaI AscI BalI BbeI BbrPI BlpI Bpu1102I

BsaAI BsaMI BsmI Bsp120I BsrGI BssHII Bst1107I BstEII BstXI ClaI CspI Csp45I

Eco47III Eco72I EcoICRI EcoNI EheI FseI HincII HindII HpaI I-PpoI KasI MluI

NaeI NarI NdeI NgoMIV PacI PflMI PinAI PmlI PpuMI PshAI Psp5II RsrII

SacI SacII SalI SgrAI SnaBI SpeI SplI SrfI Sse8387I SwaI XbaI XcmI

Table 3. Restriction Enzymes That Cut the psiCHECK?-1 Vector 6 or More Times. AciI AluI Alw26I BbvI BsaJI Bsp1286I BsrI BsrSI Bst71I

BstOI BstUI CfoI DdeI DpnI DpnII Fnu4HI FokI HaeIII

HhaI HinfI HpaII HphI Hsp92II MaeI MaeII MaeIII MboI

MboII MnlI MseI MspI NdeII NlaIII NlaIV PleI RsaI

Sau3AI Sau96I ScrFI SfaNI TaqI Tru9I XhoII

Note:The enzymes listed in boldface type are available from Promega.

Enzyme # of Sites Location

NspI 2152, 224PaeR7I 11643PmeI 11663

Ppu10I 3150, 222, 909PspAI 11649PstI 1462

PvuI 21640, 2292PvuII 180

ScaI 2662, 2180SfiI 1357SgfI 11640

SinI 2

2297, 2519

Enzyme # of Sites Location SmaI 11651SphI 2152, 224SspI 11856StuI 1403

StyI 515, 311, 404, 692,701TfiI 2426, 805Tth111I 11390VspI 12486XhoI 11643XmaI 11649

XmnI

2

1228, 2061

Table 1. Restriction Enzymes That Cut the psiCHECK?-1 Vector Between 1 and 5 Times (continued).

5.B.Restriction Enzyme Sites for the psiCHECK?-2 Vector

The following restriction enzyme tables were constructed using DNASTAR ?sequence analysis software. Please note that we have not verified this information by restriction digestion with each enzyme listed. The location given specifies the 3′-end of the cut DNA (the base to the left of the cut site).For more information on the cut sites of these enzymes, or to report a

discrepancy, please contact your local Promega Branch or Distributor. In the U.S., contact Promega Technical Services at 800-356-9526. Vector sequences are available from the GenBank ?database (GenBank ?/EMBL accession number AY535007) and online at:

https://www.sodocs.net/doc/522437431.html,/vectors/

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Enzyme # of Sites Location AatII 11391

AccI 22079, 3132Acc65I 154

AflII 4452, 649 , 1773,1897AflIII 12450

Alw44I 24702, 5948AlwNI 22094, 5853ApaI 12562AvrII 2404, 2059

BalI 31865, 3513, 4038BamHI 14451

BanII 5759, 899, 1650,2050, 2562

BbeI 42030, 2815, 3481,3613BbsI 2560, 1743BbuI 2152, 224

BclI 5734, 1187, 3112,3853, 4147BglII 11

BsaI 4514, 1234, 2123,5308

BsaAI 22083, 3734

BsaBI 41453, 2979, 4146,4450

BsaMI 32504, 4270, 4363BsmI 32504, 4270, 4363Bsp120I 12558

BspHI 33115, 4534, 5542BspMI

2

476, 3463

Enzyme # of Sites Location BsrGI 13022BssHII 11978

BssSI 33459, 4705, 6089Bst1107I 12080

Bst98I 4452, 649, 1773, 1897BstXI 13650

BstZI 31674, 4202, 4206Bsu36I 3 1280, 3145, 3745ClaI 14444Csp45I 12390

DraI 51663, 4410, 4796,5488, 5507DraIII 1882

DrdI 2441, 6160

EagI 31674, 4202, 4206EarI 51193, 1874, 2616,2727, 4575EclHKI 15374Eco47III 13519

Eco52I 31674, 4202, 4206Eco81I 31280, 3145, 3745EcoNI 32721, 3144, 4149EcoRI 21654, 2386EcoRV 11179

EheI 42028, 2813, 3479,3611

FseI 23943, 4208FspI 38, 3354, 5151HincII 14349HindII

1

4349

Table 4. Restriction Enzymes That Cut the psiCHECK?-2 Vector Between 1 and 5 Times.

5.B.

Restriction Enzyme Sites for the psiCHECK?-2 Vector (continued)Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

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Table 5. Restriction Enzymes That Do Not Cut the psiCHECK?-2 Vector.AccB7I AccIII AgeI AscI BbrPI BlpI

Bpu1102I BstEII CspI Eco72I EcoICRI I-PpoI

NdeI PacI PflMI PinAI PmlI PshAI

RsrII SacI SalI SgrAI SnaBI SpeI

SplI SrfI Sse8387I SwaI XcmI

Enzyme # of Sites Location HindIII 2420, 2497HpaI 14349

KasI 42026, 2811, 3477,3609KpnI 158MluI 12450

NaeI 33941, 3962, 4206NarI 42027, 2812, 3478,3610

NcoI 515, 311, 692, 2067, 2530

NgoMIV 33939, 3960, 4204NheI 1684NotI 11674NruI 11355

NsiI 3154, 226, 913NspI 5152, 224, 2336, 3023, 3278PaeR7I 11643PmeI 11663

Ppu10I

3

150, 222, 909

Enzyme # of Sites Location

PpuMI 12056Psp5II 12056

PspAI 21649, 2019PvuI 21640, 5005PvuII 380, 2268, 2606SacII 12036

ScaI 3662, 2697 ,4893SfiI 1357SgfI 11640

SmaI 21651, 2021SphI 2152, 224SspI 14569StuI 1403TfiI 2426, 805Tth111I 11390VspI 15199XbaI 14189XhoI 11643

XmaI 21649, 2019XmnI 2

1228, 4774

Table 4. Restriction Enzymes That Cut the psiCHECK?-2 Vector Between 1 and 5 Times (continued).

6.siCHECK? Vector Backbones and Components

The vector backbones of the psiCHECK?-1 and psiCHECK?-2 Vectors are based on the phRL-SV40 Vector (Cat.# E6261). Both the psiCHECK?-1 Vector and psiCHECK?-2 Vector contain the synthetic Renilla luciferase reporter gene.The psiCHECK?-2 Vector also contains a synthetic firefly luciferase gene.These synthetic luciferase genes have been codon optimized for more efficient mammalian expression and have been designed with a greatly reduced number of consensus transcription factor binding sites for reduced risk of anomalous transcriptional behavior.SV40 Early Enhancer/Promoter

The psiCHECK?-1 Vector and psiCHECK?-2 Vector contain the SV40 early enhancer/promoter region, which provides strong, constitutive expression of Renilla luciferase in a variety of cell types.Chimeric Intron

Downstream of the SV40 enhancer/promoter region is a chimeric intron

composed of the 5′-donor site from the first intron of the human β-globin and the branch and 3′-acceptor site from the intron that is between the leader and the body of an immunoglobin gene heavy chain variable region (22). The sequences of the donor and acceptor sites, along with the branch point site,have been changed to match the consensus sequence for splicing (23).

Transfection studies have demonstrated that the presence of an intron flanking

the cDNA insert frequently increases the level of gene expression (24–27).

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Table 6. Restriction Enzymes That Cut the psiCHECK?-2 Vector 6 or More Times. AciI AcyI AluI Alw26I AspHI AvaI AvaII BanI BbvI BglI BsaOI BsaHI BsaJI

Bsp1286I BsrI Bsr SI Bst71I BstOI BstUI CfoI Cfr10I DdeI DpnI DpnII DraII DsaI

EaeI Fnu4HI FokI HaeII HaeIII HgaI HhaI HinfI HpaII HphI Hsp92I Hsp92II MaeI

MaeII MaeIII MboI MboII MnlI MseI MspI MspA1I NciI NdeII NlaIII NlaIV PleI

PstI RsaI Sau3AI Sau96I ScrFI SfaNI SinI StyI TaqI Tru9I XhoII

Note:The enzymes listed in boldface type are available from Promega.

T7 Promoter

A T7 RNA polymerase promoter is located downstream of the chimeric intron

and immediately precedes the synthetic Renilla luciferase reporter gene. This

promoter can be used to synthesize RNA transcripts in vitro using T7 RNA

Polymerase (Cat.# P2075). Note that the T7 promoter has been verified by

sequence only; there has been no functional testing of the T7 promoter.

Polyadenylation Signals (SV40 Late and Synthetic)

Polyadenylation signals are coupled to the termination of transcription by

RNA polymerase II and signal the addition of approximately 200–250 adenosine

residues to the 3′-end of the RNA transcript (28). Polyadenylation has been

shown to enhance RNA stability and translation (29,30). The late SV40

polyadenylation signal is extremely efficient and has been shown to increase

the steady-state level of RNA to approximately fivefold more than that of the

early SV40 polyadenylation signal (31). The synthetic poly(A) was cloned from

our pCI-neo Vector (Cat.# E1841). The synthetic poly(A) signal is based on the

highly efficient polyadenylation signal of the rabbit β-globin gene (32).

7.References

1.Kumar, R., Conklin, D.S. and Mittal, V. (2003) High-throughput selection of effective

RNAi probes for gene silencing. Genome Res.13, 2333–40.

2.Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell101, 235–8.

3.Zamore, P.D. (2001) RNA interference: Listening to the sound of silence. Nature Struct.

Biol.8, 746–50.

4.Sharp, P.A. (2001) RNA interference—2001. Genes Dev.15, 485–90.

5.Gil, J. and Esteban, M. (2000) Induction of apoptosis by the dsRNA-dependent

protein kinase (PKR): Mechanism of action. Apoptosis5, 107–14.

6.Marcus, P.I. and Sekellick, M.J. (1985) Interferon induction by viruses. XIII. Detection

and assay of interferon induction-suppressing particles. Virology142, 411–5.

7.Elbashir, S.M. et al.(2001) Duplexes of 21-nucleotide RNAs mediate RNA interference

in cultured mammalian cells. Nature411, 494–8.

8.Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stable

expression of short interfering RNAs in mammalian cell. Science296, 550–3.

9.Elbashir, S.M. et al. (2002) Analysis of gene function in somatic mammalian cells

using small interfering RNAs. Methods26, 199–213.

10.Paddison, P.J. et al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific

silencing in mammalian cells. Genes Dev.16, 948–58.

11.Paul, C.P. et al.(2002) Effective expression of small interfering RNA in human cells.

Nature Biotechnol. 20, 505–8.

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

·https://www.sodocs.net/doc/522437431.html,

12.Sui, G. et al. (2002) A DNA vector-based RNAi technology to suppress gene

expression in mammalian cells. Proc. Natl. Acad. Sci. USA99, 5515–20.

13.Holen, T. et al. (2002) Positional effects of short interfering RNAs targeting the human

coagulation trigger tissue factor. Nucl. Acids Res.30, 1757–66.

14.Lee, N.S. et al. (2002) Expression of small interfering RNAs targeted against HIV-1 rev

transcripts in human cells. Nature Biotechnol. 20, 500–5.

15.Yu, J-Y., DeRuiter, S.L. and Turner, D.L. (2002) RNA interference by expression of

short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci.

USA99, 6047–52.

16.Kapadia, S.B., Brideau-Andersen, A. and Chisari, F.V. (2003) Interference of hepatitis C

virus RNA replication by short interfering RNAs. Proc. Natl. Acad. Sci. USA100, 2014–8.

17.McManus, M.T. et al.(2002) Gene silencing using micro-RNA designed hairpins. RNA

8, 842–50.

18.Hohjoh, H. (2002) RNA interference (RNA(i)) induction with various types of

synthetic oligonucleotide duplexes in cultured human cells. FEBS Lett.521, 195–9.

19.EnduRen? Live Cell Substrate Technical Manual, #TM244, Promega Corporation.

20.CellTiter-Glo?Luminescent Cell Viability Assay Technical Bulletin, #TB288, Promega

Corporation.

21.Dual-Luciferase?Reporter 1000 Assay System Technical Manual,#TM046, Promega

Corporation.

22.Bothwell, A.L. et al.(1981) Heavy chain variable region contribution to the NPb

family of antibodies: Somatic mutation evident in a gamma 2a variable region. Cell

24, 625–37.

23.Senapathy, P., Shapiro, M.B. and Harris, N.L. (1990) Splice junctions, branch point

sites, and exons: Sequence statistics, identification, and applications to genome

project. Methods Enzymol.183, 252–78.

24.Gross, M.K., Kainz, M.S. and Merrill, G.F. (1987) Introns are inconsequential to

efficient formation of cellular thymidine kinase mRNA in mouse L cells. Mol. Cell.

Biol.7, 4576–81.

25.Buchman, A.R. and Berg, P. (1988) Comparison of intron-dependent and intron-

independent gene expression. Mol. Cell. Biol.8, 4395–405.

26.Evans, M.J. and Scarpulla, R.C. (1989) Introns in the 3′-untranslated region can inhibit

chimeric CAT and beta-galactosidase gene expression. Gene84, 135–42.

27.Huang, M.T. and Gorman, C.M. (1990) Intervening sequences increase efficiency of

RNA 3′ processing and accumulation of cytoplasmic RNA. Nucl Acids Res.18, 937–47.

28.Proudfoot, N. (1991) Poly(A) signals. Cell64, 671–4.

29.Bernstein, P. and Ross, J. (1989) Poly(A), poly(A) binding protein and the regulation

of mRNA stability. Trends Biochem. Sci.14, 373–7.

30.Jackson, R.J. and Standart, N. (1990) Do the poly(A) tail and 3′ untranslated region

control mRNA translation? Cell62, 15–24.

31.Carswell, S. and Alwine, J.C. (1989) Efficiency of utilization of the simian virus 40 late

polyadenylation site: Effects of upstream sequences. Mol. Cell. Biol.9, 4248–58.

32.Levitt, N. et al.(1989) Definition of an efficient poly(A) site. Genes Dev.3, 1019–25. Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Fax 608-277-2516 ·https://www.sodocs.net/doc/522437431.html,

8.Related Products

RNA Interference Products

Product Size Cat.# GeneClip? U1 Hairpin Cloning System—Basic 1 system C8750 GeneClip? U1 Hairpin Cloning System—Puromycin 1 system C8760 GeneClip? U1 Hairpin Cloning System—Hygromycin 1 system C8770 GeneClip? U1 Hairpin Cloning System—Neomycin 1 system C8780 GeneClip? U1 Hairpin Cloning System—hMGFP 1 system C8790 Firefly and Renilla Luciferase Reagents

Product Size Cat.# EnduRen? Live Cell Substrate0.34mg E6481

3.4mg E6482

34mg E6485 Passive Lysis 5X Buffer30ml E1941 Glo Lysis Buffer, 1X100ml E2661

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

·https://www.sodocs.net/doc/522437431.html,

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

Fax 608-277-2516 ·https://www.sodocs.net/doc/522437431.html,

(a)Patent Pending.

(b)For research use only. This product and/or its use is subject to one or more of the following Promega patents: U.S. Pat.

Appln. Ser. Nos. 09/645,706, 10/943,508, 10/664,341, PCT Pat. Appln. Ser. No. PCT/US03/28939, U.S. Pat. Nos. 6,387,675 and 6,552,179, Australian Pat. No. 698424 and various corresponding patent applications and issued patents. The terms of the limited license conveyed with the purchase of this product are as follows: Researchers may use this product in their research and they may transfer derivatives to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and the recipients agree to be bound by the terms and conditions of this label license. In addition,researchers must do one of the following: (1) use luminescent assay reagents purchased from Promega Corporation for all determinations of luminescence activity resulting from the research use of this product and its derivatives; or (2) contact Promega to obtain a license for the use of the product and its derivatives in conjunction with luminescent assay reagents not purchased from Promega. No reach-through payments shall be owed to Promega relating to an organization’s

commercialization of products that are the discoveries resulting from the research use of this product or its derivatives,

provided that such products of the organization do not fall within the scope of the valid claims of any issued patents assigned or licensed to Promega, or that such commercialization would not be a violation of the terms of this label license. No other use of this product or its derivatives is authorized without the express written consent of Promega including, without

limitation, Commercial Use. Commercial Use means any and all uses of this product and derivatives by a party for monetary or other consideration and may include but is not limited to use in: (1) product manufacture; and (2) to provide a service,information or data; and/or resale of the product or its derivatives, whether or not such product or derivatives are resold for use in research. With respect to such Commercial Use, or any diagnostic, therapeutic or prophylactic uses, please contact Promega for supply and licensing information. If the purchaser is not willing to accept the conditions of this limited use statement, Promega is willing to accept the return of the unopened product and provide the purchaser with a full refund.However, in the event the product is opened, then the purchaser agrees to be bound by the conditions of this limited use statement.

(c)Licensed from University of Georgia Research Foundation, Inc., under U.S. Pat. Nos. 5,292,658, 5,418,155, Canadian Pat. No.

2,105,984 and related patents.

(d)Certain applications of this product may require licenses from others.

(e)U.S. Pat. No. 5,670,356 has been issued to Promega Corporation for a modified luciferase technology.

(f)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.

A license (from Promega for research reagent products and from The Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product.? 2002, 2004, 2005, 2007, 2009 Promega Corporation. All Rights Reserved.

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欧米茄手表真假鉴别方法

目录 前言 (1) 一、欧米茄手表真假鉴别方法 (2) 前言 海外代购自08年以来异军突起,如今已成为电子商务的的主力军之一,今后代购的发展,不再只是单纯的价格战服务、信誉、品质将成为新的行业指标。服务已成为电子商务发展的至关重要环节,一个优秀的海外代购网站,需要有统一完善的代购服务体系,通过优质的电子商务平台、国外分公司、国际快递合作配送中心、专业客服人员组成的服务中心,全程为消费者提供售前咨询、订单追踪、售后支持,能够真正让中国消费者买到优惠的国外商品,享受高品质的时尚生活。然而目前代购网站鱼龙混杂,一些低质的代购站点经常出现欺骗客户以及诽谤竞争对手的现象,导致有些刚接触该行业的用户遭遇精神和金钱上的伤害。特编辑代购相关的注意事项,以及代购相关的帮助文档,让用户真正了解代购这一行业,并且从中获益。

一、代购一般流程图解 来源:美国购物网(https://www.sodocs.net/doc/522437431.html,)"(美国购物网站|美国代购网站|代购第一门户)如需转载请注明来源:美国购物网(https://www.sodocs.net/doc/522437431.html,)"! 欧米茄是世界著名的瑞士手表,Omega手表的各个系列无论是男表还是女表都很受关注,欧米茄的超霸系列男表设计霸气十足,阳刚大气,非常适合大男生日常佩戴。女士腕表欧米茄蝶飞系列(De Ville)人气最高,属于内涵文艺设计路线,此系列腕表设计优雅奢华,适合正式场合佩戴。下面给大家分享下欧米茄手表真假鉴别方法 1.看表壳的生产序号 欧米茄手表都有生产序号,而且是一表一号,也是唯一的,通常被刻印在手表的后盖上,或

者是在手表某个壳爪的背后,一个8位的数字。如果是机械手表的话,那个8位数字的生产序号也会出现在机芯夹板的边缘上,表壳的生产序号和机芯的是一致的。 2.欧米茄手表真假鉴别方法 3.手表机芯辨别 正品手表机芯内的夹板或摆铊上标有相应的商标标牌字样;机芯在表壳组件中稳固;假冒手表机芯的夹板或摆铊上无商标标牌字样,或商标标牌字迹粗糙、模糊、歪斜,或简单地用小铜片粘贴;有些机芯内有杂志。 4.销售价格的识别 欧米茄价位均在1000元以上,高达几万元。美国代购的欧米茄手表会比专柜价格优惠一些,大概是专柜的6折到7折。

高纯度质粒小量快速提取试剂盒操作方法及步骤说明书

杭州昊鑫生物科技股份有限公司 htpp://https://www.sodocs.net/doc/522437431.html, HighPure Plasmid Mini Kit 高纯质粒小量快速提取试剂盒 目录号:PL03 试剂盒组成、储存、稳定性: 试剂盒组成保存 50次 (PL0301) 100次 (PL0302) 200次 (PL0303) 平衡液室温5ml 10ml 20ml RNaseA(10mg/ml)-20℃150μl 250μl 500μl 溶液P1 4℃15 ml 25 ml 50 ml 溶液P2 室温15 ml 25 ml 50 ml 溶液P3 室温20 ml 35 ml 70 ml 去蛋白液PE 室温16ml 31.5 ml 63 ml 第一次使用前按说明加指定量乙醇 漂洗液WB 室温15 ml 25ml 50ml 第一次使用前按说明加指定量乙醇 洗脱缓冲液EB 室温10ml 15ml 20ml 吸附柱AC 室温50个100个200个 收集管(2ml)室温50个100个200个 本试剂盒在室温储存12个月不影响使用效果。 储存事项: 1.第一次使用时,将试剂盒所带的全部RNase A加入溶液P1后(终浓度100ug/ml) 置于2-8℃保存。如果溶液P1中RNase A失活,提取的质粒可能会有微量RNA 残留,在溶液P1中补加RNase A即可。 2.环境温度低时溶液P2中SDS可能会析出浑浊或者沉淀,可在37℃水浴加热几分 钟,即可恢复澄清,不要剧烈摇晃,以免形成过量的泡沫。 3.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化,各溶液使用后应及时 盖紧盖子。 产品介绍:

本试剂盒采用改进SDS-碱裂解法裂解细胞,离心吸附柱内的硅基质膜在高盐、低pH值状态下选择性地结合溶液中的质粒DNA,再通过去蛋白液和漂洗液将杂质和其它细菌成分去除,最后低盐、高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。 产品特点: 1.离心吸附柱内硅基质膜全部采用进口世界著名公司特制吸附膜,柱与柱之间吸附 量差异极小,可重复性好。克服了国产试剂盒膜质量不稳定的弊端。 2.独有的去蛋白液配方,可以高效去除残留的核酸酶,即使是核酸酶含量丰富的菌 株如JM系列、HB101也可以轻松去除。有效防止了质粒被核酸酶降解。 3.快速、方便,不需要使用有毒的苯酚、氯仿等试剂,也不需要乙醇沉淀。获得的 质粒产量高、纯度好,可以直接用于酶切、转化、PCR、体外转录、测序等各种分子生物学实验。 注意事项 1. 所有的离心步骤均在室温完成,使用转速可以达到13,000rpm的传统台式离心机, 如Eppendorf 5415C 或者类似离心机。 2. 提取质粒的量与细菌培养浓度、质粒拷贝数等因素有关。一般高拷贝质粒,建议 接种单菌落于1.5-4.5 ml加合适抗生素的LB培养基,过夜培养14-16个小时,可提取出多达20μg的纯净质粒。如果所提质粒为低拷贝质粒或大于10kb的大质粒,应适当加大菌体使用量,使用5-10 ml过夜培养物,同时按比例增加P1、P2、P3的用量,其它步骤相同。 3. 得到的质粒DNA可用琼脂糖凝胶电泳和紫外分光光度计检测浓度与纯度。OD260 值为1相当于大约50μg/ml DNA。电泳可能为单一条带,也可能为2条或者多条DNA条带,这主要是不同程度的超螺旋构象质粒泳动位置不一造成,与提取物培养时间长短、提取时操作剧烈程度等有关。本公司产品正常操作情况下基本超螺旋可以超过90%。 4. 质粒DNA确切分子大小,必须酶切线性化后,对比DNA分子量Marker才可以知 道。处于环状或者超螺旋状态的的质粒,泳动位置不确定,无法通过电泳知道其确切大小。 5. 洗脱液EB不含有螯合剂EDTA,不影响下游酶切、连接等反应。也可以使用水洗 脱,但应该确保pH大于7.5,pH过低影响洗脱效率。用水洗脱质粒应该保存在-

专升本药学选择题

专升本药学选择题 本题答案:c 第2题下列哪种片剂不宜用硬脂酸镁作润滑剂 本题答案:c 第3题专门治理的药品是指 本题答案:d 第4题湿法制粒工艺流程图为 本题答案:b 第5题用以补充体内水分及电解质的输液是 本题答案:e 第6题舌下片应符合以下哪一条要求 本题答案:D 第7题调配处方时,如发觉处方书写不符合要求或有差错,药剂人员的正确做法是 本题答案:A

第8题关于医院制剂叙述错误的是 本题答案:A 第9题药材含水量一样为多少,大量生产浸出制剂应结合生产体会,定出含水量的操纵标准()。 A.8%~lO% B.3%~5% C.9%~16% D.12%~20% E.30%~50% 本题答案:C 第10题欲称取0.1g药物,按规定其相对误差不超过±10%,选用天平的分度值为 本题答案:C 第11题某药物的组织结合率专门低,讲明 本题答案:D 第12题非处方药分为甲、乙两类的按照是 本题答案:D 第13题西药或中成药处方,每张处方不得超过 本题答案:B

第14题口服缓释制剂可采纳的制备方法是 本题答案:D 第15题PVP是指 本题答案:B 第16题我国的药品质量监督治理的原则不包括 本题答案:C 第17题OTC分为甲、乙两类的要紧依据是 本题答案:D 第18题《药品治理法》规定,医疗机构配制制剂必须取得 本题答案:E 第19题禁止公布广告的药品是 本题答案:C 第20题甲氧氯普胺增加对乙酰氨基酚的吸取速度,其机制是本题答案:A

第21题栓剂的吸取途径中通过肝脏的首过作用的是 本题答案:A 第22题仅供皮肤使用的液体剂型是 本题答案:E 第23题关于纳米载药系统叙述错误的是 本题答案:A 第24题《药品治理法实施条例》规定,个人设置的门诊部、诊所等医疗机构不得 本题答案:A 第25题乳剂中,若水为分散相,油为连续相,则会制成何类型的乳剂 本题答案:B 第26题《医疗机构制剂许可证》的有效期为 本题答案:D 第27题我国药品技术监督的最高机构是 本题答案:D

购买欧米茄表入门指南

买表,买的是心头好。那好字就有多解了,要自己喜欢,要价格合适,要购买经过愉快无惊险,还要在使用过程中不给自己带来种种烦恼。每一样都要做到舒服,可就不容易了。那么,购买一块欧米茄手表如何才能做到一个“好”字?就要在这里说说我的见解了。 首先买表的心态要借用迷胡大师的一句话:“手表无贵贱,人心有高低”!买表难免会从品牌因素、知名度来考虑。表是自己戴的,但同时也是戴给别人看的,这一点我也无法免俗。但有一点觉得还是要想明白,手表只是市场定位差别,在真正的功能用途上是没有区别的。即使在宣传上出现了很多技术优势的手表,其实在手表技术上和其它的手表还是大同的,所存在的小异,算是特点,并无高下之分(同时代的东西在今天的科技背景下更多的是依靠专利,而不是技术优势)。 那么在买表前调整了心态,接着就应该了解一下品牌。欧米茄的历史去百度查比我码字要容易,但还是简要说些我了解的细节把。欧米茄在瑞士表中算是一个有历史、有技术的牌子。从百年前的19令机芯开始,到30mm(30T)机芯,到登月辉煌的321机芯,到全自动时代的翘楚561机芯(这个全自动时代从470、490机芯一直到752双历机芯结束),欧米茄一直都是瑞士表中的优秀品牌。可以这样说一直到70年代末期,欧米茄跟劳力士、真利时一直是瑞士表的中流砥柱,无论是技术还是销量上,都是主力军级别的(二十多年无数的天文台证书、市场占有率和好评都纪录了这个辉煌的时代)。80、90年代是欧米茄的衰败时期,随着1020机芯的停产,这样一个百年老厂在十几年的时间里面依靠这ETA机芯支撑着手表的内在,显然这样是不行的。也就是在这个时期欧米茄被劳力士拉开了品牌的距离。直至今日,欧米茄的复兴之路还没有完成,8500机芯的全面使用和9300机芯的出现只是复兴大业的第一步,未来的路还是很漫长的,但可以看见的是欧米茄又重新跻身一流手表的行列。 欧米茄现在使用的机芯在大三针上,主力是2500机芯和8500机芯(当然还有女款以及一些变型机芯,例如2202、2628之类的)。其中争议最大的2500机芯,作为欧米茄重回自产机芯的第一款产品,坊间的评价一直不佳,偷停一直是2500机芯挥之不去的阴影。个人评价,2500机芯的偷停是客观存在的,由于基础机芯ETA2892(1120)的设计取向(偏小和薄),在欧米茄做改造的时候无法大动干戈(这个是正常的,在机械上渐改永远比新设计要合适),因此遗留的问题也使欧米茄头疼。今时今日欧米茄全面启用8500机芯,开始放弃2500机芯,此项问题正是其因之一。但2500机芯并不是一只不可取的机芯,从价格体系上2500机芯是有很大优势的,全钢表2W左右的实际入手价格,在今天物价飞涨的时代已是不易。那么在买2500机芯的欧米茄表时如何避免偷停带来的烦恼呢?个人的看法是保养的注意和使用方法的注意。由于基础机芯的动力偏小,2500机芯的表要注意保持动力,每天戴满8个小时以及适当的手动补链是保证手表正常运行的基础把;减少戴戴停停的次数,不戴的时候用手动上链来保持动力;其次就是保养的间隔,2500机芯的手表正常的保养间隔5年一次的洗油个人认为似乎有缩短的必要,在保养时要尽量避免落入庸手,保证洗油的用油质量(现在看来由于基础机芯的选择似乎不当,同轴擒纵技术的高间隔保养时间反而无法实现,走反了!)。现在随着2500机芯的停产慢慢接近,过几年再想买块2W左右的欧米茄也不容易了,因此2500机芯还是属于可以放心购买的产品。最近2500D机芯开始进入市场了,这个算是大改的新型号2500机芯其实本质上是将机芯结构尽量向8500机芯靠拢,使用了三层擒纵轮,以保证擒纵系统不容易因为动力问题打滑,使机芯不再出现偷停的问题。现在看来2500D机芯的推出,将会很大程度延长2500机芯的寿命,可能还会长期作为中、低端的欧米茄入门级别手表的机芯。其实2500机芯到了D款也脱离了当初推出的时候的基础+添加的味道,开始变成真正的自产机芯。

(完整版)OMEGAdna提取说明书

材料与仪器:离心机(至少14000g)、1.5ml或2ml无核酸酶离心管、水浴锅、无水乙醇、氯仿、异戊醇、无酶水。 试验之前:先用absolute ethanol稀释DNA Wash Buffer Concentrate,每瓶DNA Wash Buffer Concentrate加80ml(200份装) 试验步骤: 1.使用mortar和pestle在liquid nitrogen中研磨样品(不超过50毫克),放入1.5ml microcentrifuge tube。 2.加350ulBuffer ML1和25ul Proteinase K,涡旋混匀,60℃孵育(最少30分钟),大多数孵育不超过4小时或者37℃孵育1晚。 3.裂解产物加350ul氯仿和异戊醇的混合溶液(24:1),涡旋混匀。10000g室温离心2min,转移上清液到1.5ml microcentrifuge tube,避免含有污染物和抑制剂的乳白色界面。 4.估计步骤3 supernatant体积,加等集体的Buffer MBL,涡旋15s混匀,70℃孵育10min。 5.加步骤3等体积的无水乙醇,涡旋15s混匀。(tips:300ul上清液+300ul Buffer MBL+300ul absolute ethanol) 6.把柱子和提供的2ml收集管装配好,加步骤5的溶液750ul,10000g 室内离心1min,倒掉被离心的液体,重复利用提供的2ml收集管。 7.把步骤5剩余的混合物按照步骤6的方法离心,但抛弃提供的2ml 收集管。 8.把柱子放在新的2ml收集管,加500ul HB Buffer,10000g,30s,第一次洗柱子。

前置过滤器使用说明书

前置过滤器使用说明书1简介 1.1 简要说明 设计标准 2简要描述 2.1 安装 2.2 使用 3安全说明 3.1简要说明 3.2电器元件 3.3发动机 3.4压力 3.5阀门 4 运输和保存 4.1 简要说明 5 安装和调试 5.1 简要说明 5.2 组成部分 5.2.1主要结构 5.2.2滤芯 5.2.3管道系统 5.2.4可动部件 5.2.5电器元件 5.3 检查 5.3.1松动的部分 5.3.2一般的方法 6操作指导 6.1 简要说明 6.2 过程描述 6.3 过程控制 6.4 短时间停机 6.5 长时间停机 6.6 检修 6.6.1具体故障 7维护和清洗 7.1 简要说明 7.1.1开启和关闭过滤器 7.1.2滤元 7.2 耐磨件 7.2.1盖子垫片 7.2.2盖子开启装置 7.3清洗 7.3.1简要说明 7.3.2过滤器箱体 7.3.3滤元 8拆除和废弃

8.1简要说明 简介 1.1 简要说明 本手册描述的是AMAFILTER凝结水过滤器的安装、使用和维护。 本装置是按照最低温度4℃以上,在室内使用,不受外界环境影响的标准设计、制造的。 本装置符合CE标准。 1.2 设计标准 本装置是为了进行液体过滤而设计的。 设计温度:60℃ 设计压力:40bar 本装置产生的音量不会超过70分贝。 简要描述 2.1 安装 过滤器是成套供货,内部元件分开包装的。安装内部元件以前,应按照正确的流向将过滤器安装于管道系统中。建议在过滤器的进出口安装压力表,这样可以通过过滤器的压降来确定运行的频率。 2.2 使用 将过滤器箱体内注满凝结水。用泵将凝结水压入滤芯,滤出液通过滤液出口离开过滤器。固体颗粒留在滤芯内或被滤料截留,这取决于使用的滤芯结构。 当滤芯两端的最大压差达到2.5bar,滤芯就失效了,需要进行更换。 经常的反洗可以提高滤芯的使用寿命。 3 安全说明 3.1 简要说明 为了避免伤害: -- 遵照当地的安全规程进行操作。 -- 在操作和维护时,应当使用一切安全设施。 -- 了解清楚装置的可动部分。 -- 在开始使用本装置时,要确认装置是处于无压状态。参照3.4节。 -- 在开启盖子的时候,要确认没有人会从盖子开启的方向过来。

袋式过滤器

目录 第一部分袋式过滤器设备的运行 1.说明 2.试运行 3.日常运行 4.过滤器设备技术性能参数表 第二部分袋式过滤器设备的维护 1.说明 2.安全问题 3.阀门 4.灰斗 5.卸灰输灰装置 6.清灰机构 7.滤袋 8.仪表 9.电气操作 第一部分袋式过滤器设备的运行 1.说明 一个性能优良的袋式过滤器,是大多数用户所期望的,但是,无论性能如何优良,如果对它的操作和维修要求了解不够、或者由责任心不强的工作人员管理的话,在短时间内也会变成性能低下的系统。同时,作为制造商来说,产品经常出现故障,不仅会不断地给业务上带来麻烦,并给人以维修费用增加、效率低下的不良印象。 另一方面,虽然选取的设备没有多少备用的能力,如果操作人员在操作与维护方面具有丰富的知识,能够很好地了解其设计上的特点,正常地进行操作与维护,就能够保持原设计的性能,充分发挥其效能,而且所需要的费用也会降到最低。 在进行设备的运转与维护时,必须按照这些说明书和资料所制定的操作规程与维护规程的规定进行工作。 为了能使袋式过滤器正确地运行,须注意以下事项: ⑴首先,用户必须选取最合适的袋式过滤器,才能降低运行与维护费用。应在定购之前,要很好地研究有关运转、测试仪表、维修等资料,再考虑合适的性能和年运行费用,来选择合适的装置。 ⑵必须按照设备制造商提供的说明书等资料中的要求进行运转。 ⑶要了解袋式过滤系统中包括那些部分。 ⑷要经常地、细致地注意滤袋的安装和工作状况。

⑸要注意进入袋式过滤器的烟气温度,一定使之在露点温度以上10℃~20℃运行。 袋式过滤系统的运行可分为:试运行和日常运行。首先,在进行试运行时,必须对系统的单一部件进行检查,然后作适应性运行,同时作一部分性能实验。其次,尽管进入了日常运行,仍然有必须经常进行检查的项目。进行这些检查对煤粉过滤器的正常运行是很有益的,尤其是在日常运行条件下,因负荷条件的变化对性能要产生一定的影响,所以先要明确操作程序,在设备投入使用后还要密切注意一段时间。 2.试运行 在新的袋式过滤器开始试运行前,必须对下列各项进行检查: ⑴风机的旋向、转速、轴承振动和温度。 ⑵管道的状况、系统的配套设备、过滤器本体是否漏气以及供水系统和供气系统等。 ⑶处理风量和各点的压力与温度是否与设计相符。 ⑷测试仪表的指示及记录是否正确。 ⑸要反复校验并确认所有安全装置都正常工作。 ⑹脉冲过滤器滤袋的检查: 滤袋在安装之初虽已调好,但在运行几天后,还必须检查滤袋的泄漏情况,因为由于温度和压力的变化、安装的问题以及反复的清灰,可能使某些滤袋的脱落现象。 ⑺新装滤袋的投运: 在开始运转的时候,常常会出现一些事先预料不到的情况,需要密切注意。例如,出现异常的温度、压力、水分等将给新装置造成损害,特别是这样的气体进入冷的过滤器时,在箱体和滤袋上可能发生水气凝结,造成滤袋堵塞和腐蚀。 另外,气体温度的急剧变化,对风机也有不良的影响,应避免这种情况。因为温度的变化,可能引起风机轴的变形,将形成不平衡状态,运行时就会引起振动。并且,在停止运行时,如温度急剧下降,再启动的时候也会有振动的危险。 设备的起动对在日常运行中保持系统的良好性能有着重要的作用,必须细心注意和慎重行事。 3、日常运行 袋式过滤器在日常运行中,由于运行条件会发生某些改变,或者出现某些故障,都将影响设备的正常运转和工作性能,要定期地进行检查和适当的调节,以尽力延长滤袋的寿命,降低运行费用,以期用最低的运行费用保持设计的最好性能。主要应注意以下一些问题。

质粒提取试剂盒-说明书-翻译

精品文档 。 1欢迎下载 E.Z.N.A.? 质粒小提试剂盒操作规程(英文版译文) (适用于No. D6942, D6943 & D6944) 1. 自新鲜划痕选择培养板中分离单菌落,接种含适当选择性抗生素的1-5ml 的LB 培 养基进行培养。37℃强力摇动(~300rpm )孵育12-16h 。使用10-20ml 培养管或容量至少4倍于培养容积的培养瓶。强烈推荐使用endA 阴性大肠杆菌菌株进行常规质粒分离。此类菌株包括DH5α和JM109。 2. 将1.5-5.0ml 细菌室温10,000 x g 离心1min 。轻轻倒出或吸走培养基并丢弃。 3. 加入250μl 的溶液I/Rnase A 重悬沉淀,涡旋或用移液器反复吹打。充分重悬沉 淀对于获得高质量的DNA 非常重要。 4. 加入250μl 溶液II ,倒置、转动试管数次使之轻轻混匀,得到透明的裂解产物。 可能需要孵育2min 。不要用力混合,以免使染色体DNA 断裂,减低质粒的纯度。该步反应不要超过5min 。溶液II 不用时要拧紧瓶盖,以免试剂被空气中的CO2酸化。 5. 加入350μl 溶液III ,立即倒置试管数次混匀,直至白色絮状沉淀物形成。为避免 形成局部沉淀,加入溶液III 后应立即、充分混匀溶液。 6. 室温≥10,000 x g 离心10分钟。白色沉淀物形成,立即进行下一步操作。 7. 小心翼翼.... 的吸取上清液,加入装配在2ml 收集管中的小量纯化柱I 中。确保离心沉淀未受扰动,确保没有细胞碎片加入到柱子中。室温下10,000 x g 离心1分钟,使裂解液完全通过柱子。 8. 丢弃滤过液,重新使用2ml 收集管;加入500μl HB 缓冲液清洗柱子,室温10,000 x g 离心1分钟,使溶液完成通过柱子。该步操作需确保残余的蛋白质污染被去除,以保证获得高品质的DNA 以适合于下游的应用。 9. 丢弃滤过液,重新使用2ml 收集管;加入700μl 用无水乙醇稀释的DNA 清洗液清 洗柱子,室温10,000 x g 离心1分钟,使溶液完全通过柱子,丢弃滤过液。 注意:DNA 清洗液的浓缩液使用前必须用无水乙醇稀释(5倍稀释),如果DNA 清洗液稀 释液经过冷藏,则使用之前必须置于室温。 10. 可选步骤:重复清洗,加入另外的700μl 用无水乙醇稀释的DNA 清洗液。 11. 将空柱子≥13,000 x g 离心2min ,使柱子的基质干燥。此步骤为关键操作,不可 遗漏。 12. 将柱子放入干净的1.5ml 微量离心管中。将30-50μl (取决于终产物的期望浓度) 洗脱液或无菌去离子水直接加入柱子基质上,使之于室温下静置1-2min 。≥13,000 x g 离心1min 洗脱DNA 。可以进行二次洗脱,以收集残存的DNA 。 13. DNA 的产量和质量:分别在波长260nm 和280nm 处测定样品适当稀释液的吸光度。DNA 的浓度计算如下: DNA 浓度=A 260×50×(稀释倍数)μg/ml A 260/A 280的比率可以反映核酸的纯度。比值大于1.8表明核算的纯度在90%以上。或者,DNA 的产量(及质量)有时可以通过琼脂糖胶/溴乙锭电泳与已知浓度的DNA 样品相比较更好的予以确定。通常情况下洗脱的大部分DNA 是超螺旋单体形式,但也可能存在串联体形式。 张小强 翻译

片剂的题目

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目录 一、产品介绍 (3) 二、工作原理 (3) 三、设备技术参数 (4) 四、售后服务承诺 (5) 五、合格证 (6)

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