MCB110L_lecture17

MCB110L_lecture17 - MCB110L Lecture 17 03/16/11 Andreas...

Info iconThis preview shows pages 1–6. Sign up to view the full content.

View Full Document Right Arrow Icon
MCB110L Lecture 17 03/16/11 Andreas Martin
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Outline: Fluorescence spectroscopy (remaining slides posted for lecture 16, 3/11/11) Experiment 7: RNA purification RT-PCR RNA quantification
Background image of page 2
Experiment 7: Gene expression analyses of Cin8 Cin8 and Kip1 with important roles in cell-cycle regulation -> gene expression profiles may depend on the activity of Cin8 mutants under different growth conditions Goal: Determine potential changes in RNA levels using RT-PCR and microarray analyses First step: RNA isolation purification of total RNA = 1 st step for many experiments in molecular biology RT-PCR and real-time PCR microarrays Northern analyses cDNA library construction in-vitro transcription/translation primer extension nuclease protection assays microarray analysis of gene expression
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Cell disruption and homogenization: critically affects yield and quality of RNA needs to be fast and thorough slow disruption may lead to -> potential RNA damage and degradation by released endogenous RNases -> changes in gene-expression patterns due to induction of transcription mechanical disruption: Dounce homogenizer, Polytron French press sonication vortexing bead milling grinding enzymatic lysis: lysozyme zymolase lysostaphin lyticase glucanase used for bacteria and yeast to degrade cell walls, capsules, coats, or capsids (hard to disrupt mechanically) followed by homogenization: sonication, vortexing different disruption methods depending on the cell type disruption and/or homogenization in lysis buffer with strong denaturants ( GdmSCN, GdmCl , SDS, phenol/chloroform)
Background image of page 4
in class: RNA purification from yeast using RNeasy Mini Kit from Qiagen: zymolase treatment of cells, lysis and homogenization in GdmSCN purification of RNA on silica-based membrane in spin columns (capacity ~ 100 μ g), RNAs < 200 bases (5.8S rRNA, 5S rRNA, tRNAs) are excluded
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 6
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 17

MCB110L_lecture17 - MCB110L Lecture 17 03/16/11 Andreas...

This preview shows document pages 1 - 6. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online