杨荣武分子生物学课件Week2modification
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IV. RNA work
h
1
What do we need DNA for?
•Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulateh
8
Plasmid purification: alkaline lysis
aqueous layer
h
5
2 ways to concentrate the genomic DNA
“spooling”
70% final conc.
Ethhanol precipitation
6
Genomic DNA prep in plants -how get rid of carbohydrates?
h
19
Inhibitors of RNase
DEPC: diethylpyrocarbonate
alkylating agent, modifying proteins and nucleic acids
fill glassware with 0.1% DEPC, let stand overnight at room temp
When is gene expressed? What is timing of gene expression? What is the level of gene expression?
(but what does an mRNA measurement really say about expression of the protein?)
CTAB:
Cationic detergent
(low ionic conditions )
h
CH3
CH3
N+ Br-
CH3
C16H33
(MC 6.61-6.62)
7
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
-- mix well, centrifuge at high speed, decant liquid
-- wash pellet (70% ethanol), dry pellet, dissolve in
appropriate volume (then determine DNA
concentration)
What about RNA?
•Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription?
h
2
DNA purification: overview
-- disulfide bridges conferring stability -- no requirement for divalent cations for activity
h
17
Common sources of RNase and how to avoid them
Contaminated solutions/buffers
h
10
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
USE GOOD STERILE TECHNIQUE TREAT SOLUTIONS WITH DEPC (when possible) MAKE SMALL BATCHES OF SOLUTIONS
Contaminated equipment
USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS BAKE GLASSWARE, 300°C, 4 hours USE “RNase-free” PIPET TIPS TREAT EQUIPMENT WITH DEPC
“cell extract”
h
4
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
solutions may be treated with DEPC -- add DEPC to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol)
h
20
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 following phenol extraction
h
13
DNA purification: overview
cell growth
cell harvest and lysis
DNA concentration
DNA purification
h
14
mRNA DNA -------------->
--------------> protein
Lots of information in mRNA:
Alkaline conditions denature DNA
Neutralize:
genomic
DNA can’t
renature
(plasmids
CAN
because
they never
fully
separate)
h
9
DNA purification: silica binding
Binding occurs in presence of high salt concentration, and is disrupted by elution with water
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
tube 4. Repeat steps 2 and 3 until there is no precipitate at
phase interface 5. (extract aqueous layer with 2 volumes of
chloroform)
h
ຫໍສະໝຸດ Baidu
12
Ethanol precipitation (DNA concentration)
• 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
h
18
Top 10 sources of RNase contamination (Ambion Scientific website)
1) Ungloved hands 2) Tips and tubes 3) Water and buffers 4) Lab surfaces 5) Endogenous cellular RNases 6) RNA samples 7) Plasmid preps 8) RNA storage (slow action of small amounts of RNAse 9) Chemical nucleases (Mg2+, Ca2+at 80°C for 5’ +) 10) Enzyme preparations
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming
h
11
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
Isolation of RNA -- Course reading 11
h
15
RNA in a typical eukaryotic cell:
10-5 micrograms RNA
80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs)
cell growth
cell harvest and lysis
DNA concentration
DNA purification
h
3
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents • Organic solvent • Proteases (lysozyme) • Heat
DNA and RNA isolation, purification, visualization and quantitation
I. Genomic DNA preparation overview
II. Plasmid DNA preparation
III. DNA purification • Phenol extraction • Ethanol precipitation
RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)
RNases are released upon cell lysis RNases are present on the skin RNases are very difficult to inactivate
About 1-5% is mRNA
-- variable in size -- but usually containing 3’ polyadenylation
h
16
The problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification
h
1
What do we need DNA for?
•Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulateh
8
Plasmid purification: alkaline lysis
aqueous layer
h
5
2 ways to concentrate the genomic DNA
“spooling”
70% final conc.
Ethhanol precipitation
6
Genomic DNA prep in plants -how get rid of carbohydrates?
h
19
Inhibitors of RNase
DEPC: diethylpyrocarbonate
alkylating agent, modifying proteins and nucleic acids
fill glassware with 0.1% DEPC, let stand overnight at room temp
When is gene expressed? What is timing of gene expression? What is the level of gene expression?
(but what does an mRNA measurement really say about expression of the protein?)
CTAB:
Cationic detergent
(low ionic conditions )
h
CH3
CH3
N+ Br-
CH3
C16H33
(MC 6.61-6.62)
7
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
-- mix well, centrifuge at high speed, decant liquid
-- wash pellet (70% ethanol), dry pellet, dissolve in
appropriate volume (then determine DNA
concentration)
What about RNA?
•Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription?
h
2
DNA purification: overview
-- disulfide bridges conferring stability -- no requirement for divalent cations for activity
h
17
Common sources of RNase and how to avoid them
Contaminated solutions/buffers
h
10
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
USE GOOD STERILE TECHNIQUE TREAT SOLUTIONS WITH DEPC (when possible) MAKE SMALL BATCHES OF SOLUTIONS
Contaminated equipment
USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS BAKE GLASSWARE, 300°C, 4 hours USE “RNase-free” PIPET TIPS TREAT EQUIPMENT WITH DEPC
“cell extract”
h
4
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
solutions may be treated with DEPC -- add DEPC to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol)
h
20
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 following phenol extraction
h
13
DNA purification: overview
cell growth
cell harvest and lysis
DNA concentration
DNA purification
h
14
mRNA DNA -------------->
--------------> protein
Lots of information in mRNA:
Alkaline conditions denature DNA
Neutralize:
genomic
DNA can’t
renature
(plasmids
CAN
because
they never
fully
separate)
h
9
DNA purification: silica binding
Binding occurs in presence of high salt concentration, and is disrupted by elution with water
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
tube 4. Repeat steps 2 and 3 until there is no precipitate at
phase interface 5. (extract aqueous layer with 2 volumes of
chloroform)
h
ຫໍສະໝຸດ Baidu
12
Ethanol precipitation (DNA concentration)
• 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
h
18
Top 10 sources of RNase contamination (Ambion Scientific website)
1) Ungloved hands 2) Tips and tubes 3) Water and buffers 4) Lab surfaces 5) Endogenous cellular RNases 6) RNA samples 7) Plasmid preps 8) RNA storage (slow action of small amounts of RNAse 9) Chemical nucleases (Mg2+, Ca2+at 80°C for 5’ +) 10) Enzyme preparations
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming
h
11
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
Isolation of RNA -- Course reading 11
h
15
RNA in a typical eukaryotic cell:
10-5 micrograms RNA
80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs)
cell growth
cell harvest and lysis
DNA concentration
DNA purification
h
3
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents • Organic solvent • Proteases (lysozyme) • Heat
DNA and RNA isolation, purification, visualization and quantitation
I. Genomic DNA preparation overview
II. Plasmid DNA preparation
III. DNA purification • Phenol extraction • Ethanol precipitation
RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)
RNases are released upon cell lysis RNases are present on the skin RNases are very difficult to inactivate
About 1-5% is mRNA
-- variable in size -- but usually containing 3’ polyadenylation
h
16
The problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification