Wako Pure Chemical
 
작성일 : 15-07-28
[Wako Pure Chemical] microRNA Cloning Kit Wako

 

microRNA Cloning Kit Wako

"High Efficiency" microRNA Cloning Kit

 

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The microRNA Cloning Kit Wako can prepare the cDNA encoding microRNA. The cloning procedure will be

completed within 1.5 days after preparation of microRNA fraction.

This kit is supported by shrimp alkaline phosphatase (SAP), thermostable single strand DNA ligase (selling

separately), and original modified adaptors.

The cloning effi ciency using this kit is improved higher than that of the conventional methods, which used

bacterial alkaline phosphatase and T4 RNA ligase.

 

Features

Kit contents (10 reactions)

High cloning effciency

• Cloning of secondary structured    microRNA

• High reproducibility of microRNA cloning

 

 

 

 

 

 

• SAP

• 5xSAP Buffer

• 40xLigation Buffer

• RNase Inhibitor

• 10mmol/L MnCl

• Reverse Transcriptase

• 10xRT Buffer

• dNTP Mixture

0.5 mol/L EDTA, pH 8.0

 

16 μL

64 μL

16 μL

16 μL

16 μL

8 μL

16 μL

112 μL

16 μL

 

  • 1mol/L Tris-HCL, pH7.5

  • Ethachinmate

  • 10mol/L Ammonium Acetate

  • 3' Adaptor (50pmol/μL)

  • 5' Adaptor(50pmol/μL)

  • RT Primer(50pmol/μL)

  • 5' PCR Primer(50pmol/μL)

  • 3' PCR Primer(50pmol/μL)

  • Control RNA(30ng/μL)

 

160 μL

24 μL

960 μL

8 μL

8 μL

8 μL

16 μL

16 μL

8 μL

 

 

Thermostable ligase and DNA polymerase are not included in this kit.

 

                         Outline of procedure

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          클릭하시면 닫힙니다.이미지 저장을 원하시면 마우스 오른쪽클릭후 '다른이름으로 저장'을 하세요
          클릭하시면 닫힙니다.이미지 저장을 원하시면 마우스 오른쪽클릭후 '다른이름으로 저장'을 하세요

 

 

Cloning of microRNA from HeLa cells

 

Procedure of microRNA cloning

 

1) Preparation of total RNA from HeLa cells (1×107 cells) by ISOGEN (Nippon Gene #315-02504, 10mL).

2) Preparation of small RNA fraction, less than 200nt, from total RNA by microRNA Isolation Kit (Bio Chain

    Institute Inc.catalog #KS341025).

3) Separation of microRNA fraction by denaturing PAGE.

4) Collection of the gels of 20~23nt region after electrophoresis.

5) Cloning by using microRNA Cloning Kit Wako (Wako catalog #290-66501).

6) Construction of the plasmids harboring cDNA encoding microRNA and transformation of E. coli.

7) Random selection of the 96 transformed E. coli from selection LB agar medium.

8) Determination and verifi cation of the cDNA sequences by using Sanger miRBase.

 

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클릭하시면 닫힙니다.이미지 저장을 원하시면 마우스 오른쪽클릭후 '다른이름으로 저장'을 하세요

            클릭하시면 닫힙니다.이미지 저장을 원하시면 마우스 오른쪽클릭후 '다른이름으로 저장'을 하세요

            클릭하시면 닫힙니다.이미지 저장을 원하시면 마우스 오른쪽클릭후 '다른이름으로 저장'을 하세요

 

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Figure. 1 Cloning efficiency of microRNA from HeLa cell lysate.

The cloning efficiency of microRNA was more than 70%. Others indicate that isolated cDNA

Sequences were not matched miRBase. Unknowns indicate that isolated cDNA sequences were not

matched human genome sequence. The contents of cloned microRNA species are indicated on Table 1.

 

 

Brochure (PDF)

 

 

Ordering informations

   

Catalog No.

Product Name

Size

292-66701

microRNA Isolation Kit, Human Ago2

10 reactions

016-20861

Anti Human AGO2, Monoclonal Antibody

50 μL

290-66501

microRNA Cloning kit wako

8 reactions

298-65103

single Standard DNA Ligase, thermostable, recombinant, Solution

200 units

292-65101

500 units

315-02504

ISOGEN

10 mL

317-02503

50 mL

311-02501

100 mL

194-13061

SuperSep15%, 12 wells

10 gels

304-30761

BioMashar1.5ml microtube (with O-ring)

50 tubes

058-07681

EasySeparator

1 set

312-01791

Ethachinmate (Nucleic acid carrier)

0.2 mL

 

 

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