第六部分 体外转录和翻译

分子生物学,教学和自学都很有效果和受益

In vitro transcription and In vitro translation

分子生物学,教学和自学都很有效果和受益

In vitro transcription

分子生物学,教学和自学都很有效果和受益

In Vitro Transcription 目的:

体外得到单一,大量的mRNA 意义:

探针------原位杂交 siRNA------RNAi Transcript------体外翻译,蛋白表达

分子生物学,教学和自学都很有效果和受益

In vitro TranscriptionEcoRI

T7BamHI

Antisense: Cut with EcoRI Use T-3 polymerase

T3

Sense: Cut with BamHI Use T-7 polymerase

分子生物学,教学和自学都很有效果和受益

分子生物学,教学和自学都很有效果和受益

分子生物学,教学和自学都很有效果和受益

Vector Maps

分子生物学,教学和自学都很有效果和受益

In vitro Transcription Plasmid with T3, T7 or SP6 promoters

Linearization of plasmid DNA by restriction

enzyme In vitro transcription: Plasmid buffer NTP labeled UTP RNA polymerase DNAse digestion, Phenol/Chloroform extraction and RNA precipitataion

分子生物学,教学和自学都很有效果和受益

In vitro transcription reactionGloves should be worn at all times during preparation of in vitro RNA. All solutions should be RNase-free i.e. made with DEPC water if home-made or bought specifically to use for RNA work.

分子生物学,教学和自学都很有效果和受益

In vitro transcription reactionYou will need:-Ribonuclease inhibitor (RNasin) -T7 or T3 RNA Polymerase -2.5 mM rNTPs -5 x T7/T3 RNA Polymerase Buffer (200mM Tris.Cl pH 8.0, 125mM NaCl, 40mM MgCl2, 10mM spermidine) -100 mM DTT -RNase-free DNase I -DEPC-treated, autoclaved

分子生物学,教学和自学都很有效果和受益

In vitro transcription reaction Linearize the plasmid at, or near, the terminus of the

insert cDNA with a suitable restriction endonuclease. Extract plasmid DNA with an equal volume of phenol/chloroform and an equal volume of chloroform prior to ethanol precipitation. Linearized plasmid DNA is resuspended in DEPCtreated, autoclaved TE, pH7.5 at a concentration of approximately 200ng/ul prior to use.

分子生物学,教学和自学都很有效果和受益

4) The standard reaction conditions are set up

containing the following quantities: -2 ul (400 ng) linearized plasmid DNA -20 U ribonuclease inhibitor -2 ul 100 mM DTT -4 ul 5 x T3/T7 RNA polymerase buffer -4 ul 2.5 mM NTPs -Sterile, DEPC treated H2O to a final volume of 19.5 ul 25 U (0.5 ul) T3/T7 RNA polymerase The reaction mixture is incubated at 37°C for 60 minutes.

分子生物学,教学和自学都很有效果和受益

DNA template is removed by the addition of 25U

RNase-free DNase I and a further incubation for 30 minutes at 37°C. The reaction mixture is phenol/chloroform extracted twice, ethanol precipitated, vacuum dried and resuspended in 25ul sterile, DEPC-treated TE, pH7.5. RNA can be analysed be electrophoresis using denaturing conditions in an agarose/formaldehyde gel system.

分子生物学,教学和自学都很有效果和受益

In Vitro Transcription 探针------原位杂交 siRNA------RNAi

Transcript------体外翻译,蛋白表达

分子生物学,教学和自学都很有效果和受益

Advantages of RNA probes: RNA-RNA hybrids are very stable Tissue can be digested with RNase (dsRNA

is not digested) after the hybridization reducing the background Higher specific activity compared to oligonucleotides

分子生物学,教学和自学都很有效果和受益

In Vitro Transcribed siRNAs In Vitro Transcribed siRNAs

- Equally effective as chemically synthesized siRNAs -Design effective siRNA Advantages -Price -Shorter turn-around time Disadvantages -More hands-on -Scalability

分子生物学,教学和自学都很有效果和受益

分子生物学,教学和自学都很有效果和受益

分子生物学,教学和自学都很有效果和受益

In vitro translation

分子生物学,教学和自学都很有效果和受益

测序技术, PCR, RT-PCR技 术,

芯片技术, RNA干扰技术

核酸信息

蛋白质功能

分子生物学,教学和自学都很有效果和受益

蛋白质功能研究思路 DNA水平缺失基因,上调下调基因的表达水

平对生命过程的影响 蛋白质水平进行研究

分离纯化蛋白质,研究其基本特性,直接在

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