BIOEN 502 Week 3
Terms
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- What is G-banding?
- chromosomes digested with trypsin and stained with Giemsa (AT rich regions dark)
- What is special karyotyping?
- chromosomes are hybridized with specific fluorescently labeled
- What does FISH do?
- Localizes a gene in the nucleus
- What are the steps of FISH?
- make nicks in DNA, place labeled probes into the nicks, denature the DNA, mix with probe DNA, and look at cells under microscope
- What is FISH used for?
- Seeing any abnormal translocations of DNA segments, can stain telomeres
- What is the Trizol method
- RNA goes into aquesous phase, DNA goes to interphase, protein to organic phase
- How is RNA gel electrophoresis done?
- Run under denaturing conditions because of secondary structure (MOPS/FOrmaldehyde)
- What are the 28S and 18S bands?
- Ribosomal rRNA visible at 2:1 intensity ratio that tell you that the RNA is good quality
- What is Northern blotting?
- RNA blotting, like southern blotting
- What is RT-PCR?
- Used for analyzing mRNA expression, two reactions: reverse transcriptase and PCR
- What is RT?
- RNA dependent DNA polymerase encoded by retroviruses
- How can RT-PCR be analyzed?
- Ethidium bromide stained gel, but it\'s not quantitative
- How do you perform real-time PCR imaging?
- Use TaqMan probes labeled with fluorophores, or SYBR green
- How do TaqMan probes work?
- 5\' exonuclease activity of the polymerase, as the FRET probe is cleaved, during syntehsis, the amount of fluorescence will increase
- How does SYBR green work for real-time PCR?
- it binds to the minor groove of DNA, fluorsecence is emitted when it is intercalated in double-stranded DNA
- How do you determine if you produced the right product through PCR? (2 ways)
- melting curve analysis, if there is only one product, there is only one peak in the derivative of the melting curve. Ct value analysis
- How do you perform absolute quantitation of real-time PCR?
- Need a standard curve, correlate fluorescence with Ct values