2012-10-19
org.kosen.entty.User@67c88bdc
정창희(tanksboy)
- 1
Plasmid에서 RNA정량 하는 방법좀 알려주세요~
Standard RNA (sRNA) preparation
The sRNA used as quantification standard wasprepared as follows: plasmid pML2G(B) (a generous gift from Dr Christos Petropoulos) was generated by
subcloning the 5 end 6·6 kb Sal I fragment of the CHO type C provirus sequence into the pUC219 vector. pML2G(B) was amplified with the reverse
primer used for the TaqMan assay and the
T7-forward primer (5-AAT TTA ATA CGA CTC ACT ATA GGG CCC CGG ACC CCT GAG TCAC-3). T7-forward primer adds a 25-base (underlined) T7 promoter sequence to the 5 end of the forward primer used for the TaqMan assay. This addi ion enables the resulting 123 bp amplification product
to be used directly as a template for sRNA synthesis
without subcloning. The obtained PCR product was
purified by agarose gel electrophoresis followed by
extraction with QIAEX II Agarose Gel Extraction
kit (Qiagen Inc., Chatsworth, CA, U.S.A.). sRNA
was prepared by in vitro transcription with
T7-MEGAshortscript kit (Ambion Inc., Austin, TX,
U.S.A.) according to the manufacturer’s instruction.
The DNA template was removed from the
sRNA preparation by RNase-free DNase treatment
immediately after the transcription.
이부분이 논문 발췌한부분이고 실험어떻게 진행하는지 이해가 안되서 올립니다.
벡터와 PCR 전문가님 좀 자세히좀 알려주세요
- plasmid
지식의 출발은 질문, 모든 지식의 완성은 답변!
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
답변 1
-
답변
백성민님의 답변
2012-10-25- 0
Vector에 있는 T7 promoter 를 포함해서 PCR product를 얻은다음 In vitro transcription을 이용해서 RNA을 얻은 것 같습니다.