All RNAs is on nitrocellulose and only one section corresponds to RNA of

All rnas is on nitrocellulose and only one section

This preview shows page 10 - 13 out of 31 pages.

All RNAs is on nitrocellulose and only one section corresponds to RNA of interest Hybridize blots with probe and wash it to get an idea where the RNA is being expressed Nucleic acids of complementary sequences can base pair to form double-stranded hybrids Overview of the RNA-seq Procedure Northern blot can only look at one gene at a time Next generation sequencing technologies look at all RNAs expressed in a given tissue at a given time Take all RNA, make cDNA, make small libraries, and do next generation sequencing Can look at quantities of each one of those cDNAs and link them to their position in the genome sequence based on the sequence that you’re finding
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You end up with RNA expression level based on nucleotide position Can say that x number of genes are expressed in x number of copies in a particular tissue at a certain time Lecture 19 Proteins: Structure, Function and Separation Strategies Some proteins have very interesting properties Make fusion proteins by linking GFP to another protein in which we are interested In doing so, you mark/label that protein and follow it in real time using a microscope Can make special vectors, reporter vectors, using GFP sequence o These reporter vectors would have GFP downstream from a promoter that you suspect might be expressed in a given tissue type o Can put that promoter in and interrogate when GFP is being expressed whenever you see green fluorescence Can make trans genes and find out when specific genes are being expressed GFP molecule was characterized structurally using X-ray diffraction and crystallization It’s composed of ribbons – beta sheets, and emits fluorescence Crystallography and X-ray diffraction have been useful to identify protein structure In order to understand the structure of a protein, you need to purify large amounts of it o Then try a different combination of salts to see if you can get crystals High concentrations of purified protein can organise to form crystal lattices These crystals can be bombarded with high energy beams (i.e. X-rays) which are scattered according to the atomic arrangement of the atoms within the crystal o Crystals are placed on a small scaffold along giant beams that fire up radiation o X-rays will hit the sample then deflect to give information how the molecules are structured relies on electron density The scatter pattern is detected by radioactive detectors and the data is analyzed by complex programs to provide a prediction of electron density “Like deciphering the shape of a rock by its ripple pattern” Electron density maps provide a skeleton upon which one can build a model By recording the electron density maps structural biologists can begin to build models by filling in the observed density with amino acids that correspond to such shapes
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Amino acids are linked into ball and stick models to complete the chain and then secondary and tertiary structures
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