Primer Extension Analysis of HBV DNA with Strand-Specific Primers
使用链特异性引物进行HBV DNA引物延伸分析
发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1552 浏览次数: 8408
评审: Smita NairVarpu MarjomakiAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Aug 2014
Advertisement
Abstract
We performed primer extension assay to determine which steps of HBV DNA synthesis (i.e., minus- and plus-strand DNA synthesis and circularization of RC DNA) are affected by phosphoacceptor site mutations in C protein. In these experiments, we used several specific oligonucleotide primers. For quantitation, the level of extended DNA (ED) was normalized to the level of a single internal standard (IS) DNA.
Materials and Reagents
- Huh7 hepatoma cells (Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB0403 )
- Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®, catalog number: 12800-017 )
- Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
- Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
- OptiMEM (Life Technologies, Gibco®, catalog number: 31985-062 )
- 500 μl Opti-MEM (Life Technologies, Gibco®, catalog number: 31985-062 )
- PEG (USB, catalog number: 19959 )
- NaCl (Sigma-Aldrich, catalog number: S3014 )
- EDTA (Sigma-Aldrich, catalog number: E5134 )
- Polyethylenimine (Polysciences, catalog number: 23966 )
- Vent Exo (-) polymerase (New England Biolabs, catalog number: M0257S )
- Micrococcal nuclease 1 μl (45 unit/μl) (Worthington Biochemical, I.U.B.: 3.1.31.1, catalog number: LS004798 )
- γ -32P-ATP (PerkinElmer Inc., catalog number: NEG035C )
- T4 polynucleotide kinase (New England Biolabs, catalog number: M0201s )
- Internal standard (IS) DNA (from HBV WT DNA Sac II/Xho I digested fragment) 1ng/1μl
- 2.5 mM dNTP mixture (Takara Bio Company, catalog number: BH7901 )
- RNase A (Fermentas, catalog number: EN05331 )
- Tris-HCl (pH 8.8) (Sigma-Aldrich, catalog number: T6066 )
- (NH4)2SO4 (Sigma-Aldrich, catalog number: T6066)
- KCl (Sigma-Aldrich, catalog number: P9541 )
- MgSO4 (Sigma-Aldrich, catalog number: 230391 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Polyacrylamide (SERVA Electrophoresis GmbH, catalog number: 10687 )
- UREA (Duksan Hi-Metal, catalog number: CAS 57-13-6 )
- APS(Sigma-Aldrich, catalog number: A3678 )
- TEMED (Sigma-Aldrich, catalog number: T9281 )
- Boric Acid(Sigma-Aldrich, catalog number: B0394 )
- EDTA (Sigma-Aldrich, catalog number: E5134)
- 1x DNA-containing reaction buffer (see Recipes)
- 5% polyacrylamide gel (see Recipes)
- 5x TBE (see Recipes)
Equipment
- 10 cm dishes (Corning Incorporated, catalog number: 430167 )
Software
- Fujifilm Image Gauge software (version 4.0)
Procedure
登录/注册后免费查看全文
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jung, J. and Kim, K. (2015). Primer Extension Analysis of HBV DNA with Strand-Specific Primers. Bio-protocol 5(15): e1552. DOI: 10.21769/BioProtoc.1552.
Download Citation in RIS Format
分类
微生物学 > 微生物生物化学 > DNA
您对这篇实验方案有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
发布问题
