Wednesday, November 08, 2006
ETHANOL PRECIPITATION OF NUCLEIC ACIDS
To ethanol precipitate nucleic acids, add salt, mix, add ethanol, mix, then freeze. Centrifuge to pellet the material.
Choice of salts:
Sodium acetate - estimate aqueous volume of DNA
Add 1/10 of this volume from a 3M sodium acetate stock
Ammonium acetate - estimate aqueous volume of DNA
Add 1/2 of this volume from a 7.5 M ammonium acetate stock.
(Final about 2.5 M, much higher than sodium cation)
""Ammonium ions help keep free nucleotides in solution.""
當你要label DNA 時, 不需用commercial column, 就可以去除free nucleotide
(請注意:如果DNA要進行Kinase反應， 則不要用ammonium acetate; 如果sample 內有SDS,建議用NaCl or NaOAc, 因為SDS 較不會沉澱)
Ethanol:
Use absolute ethanol (i.e. 200 proof = 100%) only.
Add 2.5 - 3 times the volume of the DNA/salt mixture. Mix well before freezing.
Freezing Conditions:
Freeze in a dry ice /ethanol bath for 20 minutes, a -70°C freezer for a minimum of 40 minutes, or a -20°C freezer overnight. The latter must be used for precipitating DNA in concentrated CsCl solutions since the CsCl salt will fall out of solution at lower temperatures.
RECIPES
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 57.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLAVE!
Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
=====================================================================================
another reference for DNA precipitation:
Concentrating Nucleic Acids
The need to concentrate DNA or RNA from dilute, aqueous solution arises quite frequently, for example, in almost all protocols for purification of nucleic acids from cells. The method most commonly used for this purpose is precipitation with ethanol or isopropanol.
Precipitation of DNA
A routine protocol for precipitating DNA entails the following simple steps:
1. Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample.
2. Add 2 volumes of ethanol and mix gently. If there is sufficient DNA in the sample, you will see a white precipitate form very rapidly.
3. Recover the DNA by centrifugation, and rinse the resulting pellet with 70% ethanol to remove residual salt.
4. Evaporate off residual ethanol and resuspend the DNA in the desired buffer to the desired concentration.
The image to the right shows samples of genomic DNA before and after addition of sodium acetate and ethanol. The precipitate became visible within a few seconds of adding ethanol. When the DNA in contained in small volumes, the procedures is usually carried out in 1.5 ml microcentrifuge tubes.
Like many procedures in molecular biology, there are several effective variations-on-a-theme for preciptating DNA from solution, and with a few exceptions, the choice of which to use is largely a matter of personal preference. The significant variables for nucleic acid precipitation are:
* Type and concentration of monovalent cation: The frequently-used sources of cations are:
o Sodium acetate at a final concentration of 0.3 M (3 M stock solution).
o Sodium chloride at a final concentration of 0.2 M (5 M stock solution).
o Ammonium acetate at a final concentration of 2.5 M (7.5 M stock solution)
o Lithium chloride at a final concentration of 0.8 M (8 M stock solution).
In some situations, one salt is preferred over another. For example, ammonium acetate should not be used if the DNA is going to be phosphorylated with polynucleotide kinase. If the DNA contains the detergent SDS, sodium chloride is the choice because it allows SDS to remain soluble in 70% ethanol.
* Ethanol versus isopropanol: Isopropanol is an effective alternative to ethanol and has the advantage of precipitating DNA at lower concentrations. Instead of mixing 2 volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.
* Time and temperature allowed for precipitation: Precipitation occurs very rapidly except when DNA content is very low (i.e. <> 2 hours on ice, the RNAs are collected by centrifugation. This method should not be used to prepare RNA for reverse transcription
Choice of salts:
Sodium acetate - estimate aqueous volume of DNA
Add 1/10 of this volume from a 3M sodium acetate stock
Ammonium acetate - estimate aqueous volume of DNA
Add 1/2 of this volume from a 7.5 M ammonium acetate stock.
(Final about 2.5 M, much higher than sodium cation)
""Ammonium ions help keep free nucleotides in solution.""
當你要label DNA 時, 不需用commercial column, 就可以去除free nucleotide
(請注意:如果DNA要進行Kinase反應， 則不要用ammonium acetate; 如果sample 內有SDS,建議用NaCl or NaOAc, 因為SDS 較不會沉澱)
Ethanol:
Use absolute ethanol (i.e. 200 proof = 100%) only.
Add 2.5 - 3 times the volume of the DNA/salt mixture. Mix well before freezing.
Freezing Conditions:
Freeze in a dry ice /ethanol bath for 20 minutes, a -70°C freezer for a minimum of 40 minutes, or a -20°C freezer overnight. The latter must be used for precipitating DNA in concentrated CsCl solutions since the CsCl salt will fall out of solution at lower temperatures.
RECIPES
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 57.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLAVE!
Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
=====================================================================================
another reference for DNA precipitation:
Concentrating Nucleic Acids
The need to concentrate DNA or RNA from dilute, aqueous solution arises quite frequently, for example, in almost all protocols for purification of nucleic acids from cells. The method most commonly used for this purpose is precipitation with ethanol or isopropanol.
Precipitation of DNA
A routine protocol for precipitating DNA entails the following simple steps:
1. Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample.
2. Add 2 volumes of ethanol and mix gently. If there is sufficient DNA in the sample, you will see a white precipitate form very rapidly.
3. Recover the DNA by centrifugation, and rinse the resulting pellet with 70% ethanol to remove residual salt.
4. Evaporate off residual ethanol and resuspend the DNA in the desired buffer to the desired concentration.
The image to the right shows samples of genomic DNA before and after addition of sodium acetate and ethanol. The precipitate became visible within a few seconds of adding ethanol. When the DNA in contained in small volumes, the procedures is usually carried out in 1.5 ml microcentrifuge tubes.
Like many procedures in molecular biology, there are several effective variations-on-a-theme for preciptating DNA from solution, and with a few exceptions, the choice of which to use is largely a matter of personal preference. The significant variables for nucleic acid precipitation are:
* Type and concentration of monovalent cation: The frequently-used sources of cations are:
o Sodium acetate at a final concentration of 0.3 M (3 M stock solution).
o Sodium chloride at a final concentration of 0.2 M (5 M stock solution).
o Ammonium acetate at a final concentration of 2.5 M (7.5 M stock solution)
o Lithium chloride at a final concentration of 0.8 M (8 M stock solution).
In some situations, one salt is preferred over another. For example, ammonium acetate should not be used if the DNA is going to be phosphorylated with polynucleotide kinase. If the DNA contains the detergent SDS, sodium chloride is the choice because it allows SDS to remain soluble in 70% ethanol.
* Ethanol versus isopropanol: Isopropanol is an effective alternative to ethanol and has the advantage of precipitating DNA at lower concentrations. Instead of mixing 2 volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.
* Time and temperature allowed for precipitation: Precipitation occurs very rapidly except when DNA content is very low (i.e. <> 2 hours on ice, the RNAs are collected by centrifugation. This method should not be used to prepare RNA for reverse transcription
Sunday, November 05, 2006
Purification of GST-Fusion Protein
Purification of GST-Fusion Protein
Materials:
PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3)
PBST: PBS + 0.5% Triton X-100
Glutathione Resin: Glutathione Sepharose 4B (Pharmacia 17-07556-01)
Elution Buffer: 100 mM Tris (pH 8.5-9.0), 20 mM reduced glutathione (aliquot and store @ -20 deg C)
0.5 M IPTG: prepare in H2O and freeze in aliquots
Protease Inhibitors: add to solutions immediately before use
Procedure:
1. Induce expression of GST-fusion protein
* grow 5 ml O/N culture of E. coli strain harboring pGEX plasmid in LB + Carb (50 ug/ml)
* innoculate 500 mls LB+Carb media with 5 mls saturated O/N culture
* grow culture at 30 degrees until mid-log phase (OD600 ~ 0.6 - 0.7)
o typically 2.5-3.5 hours for TG1 host strains
o collect 100 ul cells for analysis and resuspend in 10 ul PBS
* induce fusion protein expression by adding IPTG to 0.1 mM
o grow cells for 3 additional hours at 30 degrees (final OD600 ~ 1.2 - 1.3)
o collect 100 ul cells for analysis and resuspend in 20 ul PBS buffer
* harvest cells by centrifugation 3000 x gav for 8 min (5K rpm in SLA-1500 rotor)
* resuspend cells in 20 mls PBS
* concentrate cells by centrifugation 3000 x g for 8 min and resuspend in final volume of PBS no greater than 5 mls
* transfer to 50 ml conical tube
2. Prepare extract (all work done @ 4 degrees C)
* freeze cells at -80 degrees 1 hr - O/N
* thaw cells and add protease inhibitor cocktail
* lyse cells by sonication: 4 - 6 x 10 seconds with 45 second rests on ice between bursts
o (Fisher 550 Sonic Dismembranator @ setting 4.5 with microtip)
o monitor cell lysis by examination with compound microscope
* add Triton X-100 to final concentration of 1% and rock gently for 20 minutes
* clarify extract by centrifugation
o transfer lysate to centrifuge tube
o wash and collect residual with 5 mls GST buffer and combine with lysate
o spin down insoluble material 10 min @ 8000 x gav (10K rpm in SS-34 rotor)
* transfer supernatant to clean tube
o save 10 ul for analysis
* resupend pellet in 10 mls PBST and save 10 ul for analysis
3. Prepare glutathione resin
* add 1.33 mls 75% glutathione sepharose slurry to 15 ml conical tube ( = 1 ml bed volume)
* spin down resin 2 min @ 500 x g (2K rpm in IEC tabletop centrifuge) and remove supernatant
* wash 2x with cold PBS
* wash 1x with cold PBST
4. Bind GST-fusion protein to GSH-resin
* transfer clarified extract to prepared glutathione resin
* rock gently @ 4 degrees for 2 hours to allow binding
* remove unbound material by centrifugation 2 min @ 500 x g and removal of supernatant
* wash resin 2x with cold PBST
* wash resin 1x with cold PBS
5. Elute bound protein from resin
* transfer resin to empty column and allow resin to settle
* elute protein from resin in 10 x 250 ul fractions
* determine fractions containing protein by Bradford assay
o GST-fusion proteins typically eluted in fractions 3-6
* asses protein quality by SDS-PAGE of protein containing fractions
* pool protein containing fractions and store at -80 degrees
Notes & Misc:
* Often a 70 kD protein co-purifies with the GST-fusion protein. This is likely the chaparonin DnaK and can sometimes be removed by treating the clarified lysate with 2 mM ATP, 10 mM MgSO4 before binding the GST-fusion to the resin.
* Use 25-30 degrees instead of 37 degrees for cultures to improve solubility of GST-fusion proteins
* 1 ml bed volume of resin is reported to bind approx 5-8 mg protein
References:
* GST Gene Fusion System (Pharmacia Biotech)
* solubilization from inclusion bodies: Frangioni, J.V. and B.G. Neel (1993). Anal. Biochem. 210:179-187.
protocol compiled by Chad Rappleye
Aroian Lab Protocols
Materials:
PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3)
PBST: PBS + 0.5% Triton X-100
Glutathione Resin: Glutathione Sepharose 4B (Pharmacia 17-07556-01)
Elution Buffer: 100 mM Tris (pH 8.5-9.0), 20 mM reduced glutathione (aliquot and store @ -20 deg C)
0.5 M IPTG: prepare in H2O and freeze in aliquots
Protease Inhibitors: add to solutions immediately before use
Procedure:
1. Induce expression of GST-fusion protein
* grow 5 ml O/N culture of E. coli strain harboring pGEX plasmid in LB + Carb (50 ug/ml)
* innoculate 500 mls LB+Carb media with 5 mls saturated O/N culture
* grow culture at 30 degrees until mid-log phase (OD600 ~ 0.6 - 0.7)
o typically 2.5-3.5 hours for TG1 host strains
o collect 100 ul cells for analysis and resuspend in 10 ul PBS
* induce fusion protein expression by adding IPTG to 0.1 mM
o grow cells for 3 additional hours at 30 degrees (final OD600 ~ 1.2 - 1.3)
o collect 100 ul cells for analysis and resuspend in 20 ul PBS buffer
* harvest cells by centrifugation 3000 x gav for 8 min (5K rpm in SLA-1500 rotor)
* resuspend cells in 20 mls PBS
* concentrate cells by centrifugation 3000 x g for 8 min and resuspend in final volume of PBS no greater than 5 mls
* transfer to 50 ml conical tube
2. Prepare extract (all work done @ 4 degrees C)
* freeze cells at -80 degrees 1 hr - O/N
* thaw cells and add protease inhibitor cocktail
* lyse cells by sonication: 4 - 6 x 10 seconds with 45 second rests on ice between bursts
o (Fisher 550 Sonic Dismembranator @ setting 4.5 with microtip)
o monitor cell lysis by examination with compound microscope
* add Triton X-100 to final concentration of 1% and rock gently for 20 minutes
* clarify extract by centrifugation
o transfer lysate to centrifuge tube
o wash and collect residual with 5 mls GST buffer and combine with lysate
o spin down insoluble material 10 min @ 8000 x gav (10K rpm in SS-34 rotor)
* transfer supernatant to clean tube
o save 10 ul for analysis
* resupend pellet in 10 mls PBST and save 10 ul for analysis
3. Prepare glutathione resin
* add 1.33 mls 75% glutathione sepharose slurry to 15 ml conical tube ( = 1 ml bed volume)
* spin down resin 2 min @ 500 x g (2K rpm in IEC tabletop centrifuge) and remove supernatant
* wash 2x with cold PBS
* wash 1x with cold PBST
4. Bind GST-fusion protein to GSH-resin
* transfer clarified extract to prepared glutathione resin
* rock gently @ 4 degrees for 2 hours to allow binding
* remove unbound material by centrifugation 2 min @ 500 x g and removal of supernatant
* wash resin 2x with cold PBST
* wash resin 1x with cold PBS
5. Elute bound protein from resin
* transfer resin to empty column and allow resin to settle
* elute protein from resin in 10 x 250 ul fractions
* determine fractions containing protein by Bradford assay
o GST-fusion proteins typically eluted in fractions 3-6
* asses protein quality by SDS-PAGE of protein containing fractions
* pool protein containing fractions and store at -80 degrees
Notes & Misc:
* Often a 70 kD protein co-purifies with the GST-fusion protein. This is likely the chaparonin DnaK and can sometimes be removed by treating the clarified lysate with 2 mM ATP, 10 mM MgSO4 before binding the GST-fusion to the resin.
* Use 25-30 degrees instead of 37 degrees for cultures to improve solubility of GST-fusion proteins
* 1 ml bed volume of resin is reported to bind approx 5-8 mg protein
References:
* GST Gene Fusion System (Pharmacia Biotech)
* solubilization from inclusion bodies: Frangioni, J.V. and B.G. Neel (1993). Anal. Biochem. 210:179-187.
protocol compiled by Chad Rappleye
Aroian Lab Protocols
hTOP2 beta Purification- Preparation of Loading samples
2. Preparation of loading samples
- thaw cells on ice or RT (10 g of cells in 10 ml of Buffer I)
- add 5 ml of acid-washed glassbeads and protease inhibitors cocktail
- vortex 20'' and cool mixture at -5oC for 40'' as one cycle, repeat vortex cycle for 10 min.
- spin lysate at 7700g for 10 min
- transfer supernant to a clean tube (about 8 mL)
- dilute the supernant by Dilution Buffer (ratio 1:4)
- high salt-PEG precipitation for 30 min on ice by mixing
- spin at 10-12,000 Xg for 60 min
- about 30 ml of supernant is the sample for loading on HAP column.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
## Dilution Buffer:
30 mM KPi (3 mL 2M KPi)
2mM MgCl2 (0.4mL 1M MgCl2)
1mM PMSF ( 2mL 0.1 M PMSF)
1 mM Beta-Mercaptoethanol (-.014 mL 14.2M beta-ME)
1mM DTT ( 0.2 mL 1M DTT)
10 % glycerol ( 20 mL gylcerol)
1M NaCl ( 50 mL 5M NaCl)
5ug/ml leupeptin (0.5mL 2 mg/mL)
5ug/ml pepstatin A (0.5mL 2mg/mL)
dH2O 103.4mL
# Dialysis Buffer: (4 Liter)
Kpi pH 7.0 30mM 60mL 2M
Glycerol 50% 2 liters
DTT 2mM 8mL 1M
EDTA 0.5mM 4mL 0.5M
PMSF 1mM 4mL
- thaw cells on ice or RT (10 g of cells in 10 ml of Buffer I)
- add 5 ml of acid-washed glassbeads and protease inhibitors cocktail
- vortex 20'' and cool mixture at -5oC for 40'' as one cycle, repeat vortex cycle for 10 min.
- spin lysate at 7700g for 10 min
- transfer supernant to a clean tube (about 8 mL)
- dilute the supernant by Dilution Buffer (ratio 1:4)
- high salt-PEG precipitation for 30 min on ice by mixing
- spin at 10-12,000 Xg for 60 min
- about 30 ml of supernant is the sample for loading on HAP column.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
## Dilution Buffer:
30 mM KPi (3 mL 2M KPi)
2mM MgCl2 (0.4mL 1M MgCl2)
1mM PMSF ( 2mL 0.1 M PMSF)
1 mM Beta-Mercaptoethanol (-.014 mL 14.2M beta-ME)
1mM DTT ( 0.2 mL 1M DTT)
10 % glycerol ( 20 mL gylcerol)
1M NaCl ( 50 mL 5M NaCl)
5ug/ml leupeptin (0.5mL 2 mg/mL)
5ug/ml pepstatin A (0.5mL 2mg/mL)
dH2O 103.4mL
# Dialysis Buffer: (4 Liter)
Kpi pH 7.0 30mM 60mL 2M
Glycerol 50% 2 liters
DTT 2mM 8mL 1M
EDTA 0.5mM 4mL 0.5M
PMSF 1mM 4mL
hTOP2 beta Purification- Preparation of Yeast cells
1. Preparation of Yeast cells
- inoculate hTOP2beta/BCY123 into 50 ml of Ura- SD medium with 2% glucose and grow at 30oC for overnight.
- dilute cell culture 1:100 to Ura- SD medium with 3% glycerol and 1% lactic acid and grow for about another 12 hours.
- when OD 600 reach about 0.75, add final 2% galacotse to the culture (for induction of Galatose promoter)
- after induction by galactose for 6-14 hrs, harvest cells by 3000rpm spin for 5 min.
-wash cells once with ice-cold water and once with Buffer I.
- resuspend cells in Buffer I 1ml per 1g of cell pellete.
-freeze cells in liquide nitrogen and store frozen cells at -70oC.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
- inoculate hTOP2beta/BCY123 into 50 ml of Ura- SD medium with 2% glucose and grow at 30oC for overnight.
- dilute cell culture 1:100 to Ura- SD medium with 3% glycerol and 1% lactic acid and grow for about another 12 hours.
- when OD 600 reach about 0.75, add final 2% galacotse to the culture (for induction of Galatose promoter)
- after induction by galactose for 6-14 hrs, harvest cells by 3000rpm spin for 5 min.
-wash cells once with ice-cold water and once with Buffer I.
- resuspend cells in Buffer I 1ml per 1g of cell pellete.
-freeze cells in liquide nitrogen and store frozen cells at -70oC.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
