基于DNA的PCR法分析免疫球蛋白重链（Igh）的重排

Yusuke  Satoh; Takao  Sudo; Takafumi  Yokota

doi:10.21769/BioProtoc.1046

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Peer-reviewed

DNA PCR Assays for Igh Rearrangement

基于DNA的PCR法分析免疫球蛋白重链（Igh）的重排

Yusuke Satoh email

TS Takao Sudo

Takafumi Yokota

发布: 2014年02月20日第4卷第4期 DOI: 10.21769/BioProtoc.1046 浏览次数: 10056

评审: Omar AkilAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Jun 2013

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Abstract

This protocol is used for the detection of immunoglobulin heavy (H) chain rearrangements. This PCR-based assay enables detection of D_H-J_H recombination in cultured hematopoietic cells (Schlissel et al., 1991; Satoh et al., 2013) [e.g. ES-derived cells (Satoh et al., 2013)].

Materials and Reagents

Mouse spleen cells or ES-derived hematopoietic cells
DNA extraction Kit: PerfectPure DNA Cultured Cell Kit (5 PRIME, catalog number: 2302000 )
10x PCR Buffer with KCl (Life Technologies, Applied Biosystems^®, catalog number: 4338856 )
MgCl₂ (Life Technologies, Applied Biosystems^®, catalog number: 4338856)
Taq DNA polymerase (Life Technologies, Applied Biosystems^®, catalog number: 4338856)
dNTPs (Life Technologies, Applied Biosystems^®, catalog number: 4338856)
Primers (FASMAC)
The sequence of primers are as follows.
a. D_HL(5’): GGAATTCG(AorC)TTTTTGT(CorG)AAGGGATCTACTACTGTG
b. Mu0(5’): CCGCATGCCAAGGCTAGCCTGAAAGATTACC
c. J3(3’): GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
Agarose (UltraPure^TM Agarose) (Life Technologies, Invitrogen^TM, catalog number: 16500-100 )
Ethidium bromide (Wako Pure Chemical Industries, catalog number: 315-90051 )

Equipment

PCR Thermal Cycler (Veriti Thermal Cycler) (Life Technologies, Applied Biosystems^®)
Centrifuges (TOMY SEIKO, model: MX-150 )
Electrophoresis apparatus (ADVANCE, Mupid-exU)

Procedure

English

中文翻译

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如何引用

Satoh, Y., Sudo, T. and Yokota, T. (2014). DNA PCR Assays for Igh Rearrangement. Bio-protocol 4(4): e1046. DOI: 10.21769/BioProtoc.1046.