杨荣武分子生物学课件Week2modification

Plasmid purification: alkaline lysis
Alkaline conditions denature DNA Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate)
2 ways to concentrate the genomic DNA
70% final conc.
“spooling”
Ethanol precipitation
Genomic DNA prep in plants -how get rid of carbohydrates?
CTAB:
Cationic detergent
cell growth
cell harvest and lysis
DNA concentration
DNA purification
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents • Organic solvent • Proteases (lysozyme) • Heat
DNA and RNA isolation, purification, visualization and quantitation
I. II. III. Genomic DNA preparation overview Plasmid DNA preparation DNA purification • Phenol extraction • Ethanol precipitation RNA work
Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming
Phenol extraction
1. Aqueous volume (at least 200 microliters)
2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube 4. Repeat steps 2 and 3 until there is no precipitate at
“cell extract”
Genomic DNA prep: removing proteins and RNA
chloroform
Need to mix gently! (to avoid shearing breakage of the genomic DNA)
Add the enzyme RNase to degrade RNA in the aqueous layer
IV.
What do we need DNA for?
•Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA)
DNA purification: silica binding
Binding 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
What about RNA?
•Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription?
DNA purification: overview
phase interface
5. (extract aqueous layer with 2 volumes of chloroform)
Ethanol precipitation (DNA concentration)
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) -- add 22 microliters sodium acetate 3M pH 5.2
CH3 (low ionic conditions) CH3 N+
CH3 Br-
C16H33
(MC 6.61-6.62)
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
pHale Waihona Puke Baiduasmids
Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate