• PDF / 183,894 Bytes
• 16 Pages / 439.37 x 666.142 pts Page_size
• 12 Downloads / 141 Views

DOWNLOAD

REPORT

1

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3996

2

Real-Time Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3998

3 Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3999 3.1 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4002 4

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4002

5 5.1 5.2 5.3 5.4 5.5 5.6 5.7

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4003 Primer and Probe Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4003 Probe (Taqman Assays Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4003 Primer Optimization (SYBR Green Assay) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4003 Standard Curve Construction from Plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4003 Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4005 TaqMan Probe Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4005 Real-Time PCR Using SYBR Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4006

6 Two-Step Reverse Transcription Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4006 6.1 Reverse Transcription (RT) Reaction Using Superscript III (Invitrogen) . . . . . . . . . . 4006 6.2 RT Real-Time PCR Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4007 7

Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4008

K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_311, # Springer-Verlag Berlin Heidelberg, 2010

3996

48

Real-Time PCR Approaches for Analysis of Hydrocarbon-Degrading Bacterial Communities

Abstract: Since the development of the polymerase chain reaction (PCR) in the 1980s our knowledge of environmental microbial diversity and function has increased greatly. However quantification of particular environmental microbes by ‘‘end-point PCR’’ techniques has typically been inaccurate due to inherent l

Recommend Documents

Coral Reef Bacterial Communities

243 82 1MB

Multiplex PCR for Identification of Bacterial Pathogens of Infectious Pneumonia

163 105 298KB

Falling bacterial communities from the atmosphere

196 90 2MB

Affine analysis for quantitative PCR measurements

170 32 4MB

Beyond Antibiotics: Photo/Sonodynamic Approaches for Bacterial Theranostics

141 92 3MB

Development of quantitative real-time PCR primers for detecting 42 oral bacterial species

143 24 421KB

Molecular Approaches to Diagnosis in Ewing Sarcoma: RT-PCR

164 51 10MB

Realtime Data Mining Self-Learning Techniques for Recommendation Eng

242 9 7MB

Quantification of Ammonia Oxidizing Bacterial Abundances in Environmental Samples by Quantitative-PCR

157 58 10MB

PCR

193 28 57MB

Error Analysis: Geometric Approaches

189 53 3MB

Pcr

184 0 1MB