實驗概要
完成這個RACE需要全套反轉錄系統，當然選一個好的反轉錄酶（無RNase H），dUTP，taq酶，Uracil DNA glycosylase ，還有這幾條引物：
Adapter A AUCUCGAGUUCGCGCCGGAUCC(T) 25 VN cDNA synthesis and adapter-A incorporation
Adapter B AUAUGCACUGCCGCGUCUGAGGGGGGGG Cap ?nder adapter-B incorporation
Primer A CUCGAGUUCGCGCCGGAUC Second-strand cDNA synthesis
Primer B AUAUGCACUGCCGCGUCUGA Second-strand cDNA synthesis
5
T-RACE primer ATATGCACTGCCGCGTCTGA 5 speci?c ampli?cation of cDNA
ends 3 T-RACE primer CTCGAGTTCGCGCCGGATC 3 speci?c ampli?cation of
cDNA ends
實驗原理
原理圖：
完成這個RACE你需要全套反轉錄系統，當然選一個好的反轉錄酶（無RNase H），dUTP，taq酶，Uracil DNA glycosylase ，還有這幾條引物：
Adapter A AUCUCGAGUUCGCGCCGGAUCC(T) 25 VN cDNA synthesis and adapter-A incorporation
Adapter B AUAUGCACUGCCGCGUCUGAGGGGGGGG Cap ?nder adapter-B incorporation
Primer A CUCGAGUUCGCGCCGGAUC Second-strand cDNA synthesis
Primer B AUAUGCACUGCCGCGUCUGA Second-strand cDNA synthesis
5
T-RACE primer ATATGCACTGCCGCGTCTGA 5 speci?c ampli?cation of cDNA
ends 3 T-RACE primer CTCGAGTTCGCGCCGGATC 3 speci?c ampli?cation of
cDNA ends
實驗步驟
開始DIY：
1、RNA extraction（推薦Trizol，具體省略）
2、First-strand cDNA synthesis incorporating dUTP and 5 and 3 adapter sequences：
The RNA, Adapter A and dNTPs were incubatedat 65 C for 5 min and then chilled on ice. Add the remaining components in the following reaction mixture (20 ml): 2.5 mM Adapter A, RT buffer, 1.0 mMdUTP, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP,0.125 mM dTTP, 40 U RNAse inhibitor, 200 U MMLV reverse transcriptase and incubated for 1h at 42 C. After incubation, 0.4 ml of fresh 100 mM MnCl2 and 1 ml of Adapter B (10 mM) were added to the reaction that was further incubated for 15 min at 42 C. Then heat inactivated at 70 C for 10 min. Degradation of the RNA in the RNA/cDNA hybrid was achieved by the addition of 1 ml RNAse H and incubation at 37 C for 15 min. The reaction was purfied through a QIAquick PCR purification column.
3、Second-strand synthesis incorporating dUTP by PCR:
Second-strand synthesis was achieved by PCR using the priming sites incorporated at the 3 (Adapter B) and 5 (Adapter A) ends of the first-strand cDNA by 21 cycles.The reaction was then chilled on ice and puri?ed through a QIAquick PCR puri?cation column.
4、Asymmetric PCR using standard dNTPs：
Asymmetric PCR was performed using a gene-specific primer (GSP1) and standard dNTPs (dATP, dTTP,dGTP, dCTP)by 20 cycles.The reaction was chilled on ice, puri?ed through a MinElute PCR puri?cation column.
5、Uracil DNA glycosylase degradation of non-target dUTP containing cDNAs：
cDNAs incorporating dUTP were removed by UDG treatment to leave only those cDNAs synthesized with standard dNTPs during the asymmetric PCR. The following 17.8 ml reaction was performed: 10 ml asymmetric PCR product, 2 ml (two units) Uracil DNA glycosylase, 2 ml 10X PCR buffer, 1.2 ml 50 mM MgCl2 , 1ml nested GSP2, 1ml of 10 mM T-RACE primer, 0.6 ml water and incubated at 37 C for 30 min.6、Targeted ampli?cation of cDNA ends (T-RACE PCR): The UDG-treated cDNA mixture was heated to 95 C before the addition of 2 ml 2.5 mM standard dNTPs and 0.2 ml (one unit) of Taq DNA polymerase. PCR by 35 cycles .
出處文章：Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences
雜志：Nucleic Acids Research, 2010, Vol. 38, No. 21 e194（影響因子8.026）
完成，不明白的請評論留言。
附 件 (共1個附件,占414KB)
Targeted rapid
414KB
下載