《杨荣武分子生物学课件week2modification》由会员分享，可在线阅读，更多相关《杨荣武分子生物学课件week2modification（58页珍藏版）》请在金锄头文库上搜索。
1、DNA and RNA isolation, purification, visualization and quantitation,Genomic DNA preparation overview Plasmid DNA preparation DNA purification Phenol extraction Ethanol precipitation RNA work,What do we need DNA for?,Detect, enumerate, clone genes Detect, enumerate species Detect/sequence specific DN
2、A regions Create new DNA “constructs” (recombinant DNA),What about RNA?,Which genes are being transcribed? When/where are genes being transcribed? What is the level of transcription?,cell growth,cell harvest and lysis,DNA purification,DNA purification: overview,DNA concentration,Bacterial genomic DN
3、A prep: cell extract,Lysis: Detergents Organic solvent Proteases (lysozyme) Heat,“cell extract”,Genomic DNA prep: removing proteins and RNA,Add the enzyme RNase to degrade RNA in the aqueous layer,Need to mix gently! (to avoid shearing breakage of the genomic DNA),chloroform,2 ways to concentrate th
4、e genomic DNA,70% final conc.,“spooling”,Ethanol precipitation,Genomic DNA prep in plants - how get rid of carbohydrates?,CTAB: Cationic detergent (MC 6.61-6.62),(low ionic conditions),N+,CH3,Br-,CH3,CH3,C16H33,Plasmids: vehicles of recombinant DNA,Bacterial cell,genomic DNA,plasmids,Non-chromosomal
5、 DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate,Plasmid purification: alkaline lysis,Alkaline conditions denature DNA Neutralize: genomic DNA cant renature (plasmids CAN because they never fully separate),DNA purification: silica binding,Bindin
6、g occurs in presence of high salt concentration, and is disrupted by elution with water,DNA purification: phenol/chloroform extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer
7、 Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming,Aqueous volume (at least 200 microliters) Add 2 volumes of phenol:chloroform, mix well Spin in centrifuge, move aqueous phase to a new tube Repeat steps 2 and 3 until there is no precipitate at phase interface (extract
8、 aqueous layer with 2 volumes of chloroform),Phenol extraction,Ethanol depletes the hydration shell surrounding DNA Allowing cations to interact with the DNA phosphates Reducing repulsive forces between DNA strands Causing aggregation and precipitation of DNA Aqueous volume (example: 200 microliters
9、) - add 22 microliters sodium acetate 3M pH 5.2 - add 1 microliter of glycogen (gives a visible pellet) - add 2 volumes (446 microliters) 100% ethanol - mix well, centrifuge at high speed, decant liquid - wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then determine DNA conce
10、ntration),Ethanol precipitation (DNA concentration),cell growth,cell harvest and lysis,DNA purification,DNA purification: overview,DNA concentration,Isolation of RNA - Course reading 11,DNA - mRNA - protein,Lots of information in mRNA: When is gene expressed? What is timing of gene expression? What
11、is the level of gene expression? (but what does an mRNA measurement really say about expression of the protein?),RNA in a typical eukaryotic cell: 10-5 micrograms RNA 80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs) About 1-5% is mRNA - variable in size - but usually containing
12、 3 polyadenylation,The problem(s) with RNA: RNA is chemically unstable - spontaneous cleavage of phosphodiester backbone via intramolecular transesterification RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases) RNases are released upon cell lysis RNases are present on the skin RN
13、ases are very difficult to inactivate - disulfide bridges conferring stability - no requirement for divalent cations for activity,Common sources of RNase and how to avoid them,Contaminated solutions/buffers USE GOOD STERILE TECHNIQUE TREAT SOLUTIONS WITH DEPC (when possible) MAKE SMALL BATCHES OF SO
14、LUTIONS Contaminated equipment USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS BAKE GLASSWARE, 300C, 4 hours USE “RNase-free” PIPET TIPS TREAT EQUIPMENT WITH DEPC,Top 10 sources of RNase contamination (Ambion Scientific website) Ungloved hands Tips and tubes Water and buffers Lab surfaces Endogenous cell
15、ular RNases RNA samples Plasmid preps RNA storage (slow action of small amounts of RNAse Chemical nucleases (Mg2+, Ca2+at 80C for 5 +) Enzyme preparations,Inhibitors of RNase DEPC: diethylpyrocarbonate alkylating agent, modifying proteins and nucleic acids fill glassware with 0.1% DEPC, let stand ov
16、ernight at room temp solutions may be treated with DEPC - add DEPC to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol),Inhibitors of Rnase Vanadyl ribonucleoside complexes competitive inhibitors of RNAses, but need to be removed from the final preparation of RNA Protein inhibitors of RNase horseshoe-shaped, leucine rich protein, found in cytoplasm of most mammalian tissues must be replenished followin
