杨荣武分子生物学
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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
“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
2 ways to concentrate the genomic DNA
“spooling”
70% final conc.
Ethanol precipitation
Genomic DNA prep in plants -how get rid of carbohydrates?
CTAB:
Cationic detergent
1:1 phenol : chloroform or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer
IV. RNA work
What do we need DNA for?
•Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA
cell growth
cell harvest and lysis
DNA concentration
DNA purification
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents • Organic solvent • Proteases (lysozyme) • Heat
Plasmid purification: alkaline lysis
Alkaline conditions denature DNA
Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate)
DNA purification: silica binding
Binding occurs in presence of high salt concentration, and is disrupted by elution with water
DNA purification: phenol/chloroform extraction
tube 4. Repeat steps 2 and 3 until there is no precipitate at
phase interface 5. (extract aqueoห้องสมุดไป่ตู้s layer with 2 volumes of
chloroform)
Ethanol precipitation (DNA concentration)
What about RNA?
•Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription?
DNA purification: overview
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
(low ionic conditions )
CH3
CH3
N+ Br-
CH3
C16H33
(MC 6.61-6.62)
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate
• Aqueous volume (example: 200 microliters) -- 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 concentration)
DNA and RNA isolation, purification, visualization and quantitation
I. Genomic DNA preparation overview
II. Plasmid DNA preparation
III. DNA purification • Phenol extraction • Ethanol precipitation
“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
2 ways to concentrate the genomic DNA
“spooling”
70% final conc.
Ethanol precipitation
Genomic DNA prep in plants -how get rid of carbohydrates?
CTAB:
Cationic detergent
1:1 phenol : chloroform or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer
IV. RNA work
What do we need DNA for?
•Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA
cell growth
cell harvest and lysis
DNA concentration
DNA purification
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents • Organic solvent • Proteases (lysozyme) • Heat
Plasmid purification: alkaline lysis
Alkaline conditions denature DNA
Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate)
DNA purification: silica binding
Binding occurs in presence of high salt concentration, and is disrupted by elution with water
DNA purification: phenol/chloroform extraction
tube 4. Repeat steps 2 and 3 until there is no precipitate at
phase interface 5. (extract aqueoห้องสมุดไป่ตู้s layer with 2 volumes of
chloroform)
Ethanol precipitation (DNA concentration)
What about RNA?
•Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription?
DNA purification: overview
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
(low ionic conditions )
CH3
CH3
N+ Br-
CH3
C16H33
(MC 6.61-6.62)
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate
• Aqueous volume (example: 200 microliters) -- 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 concentration)
DNA and RNA isolation, purification, visualization and quantitation
I. Genomic DNA preparation overview
II. Plasmid DNA preparation
III. DNA purification • Phenol extraction • Ethanol precipitation