Preparation of DNA affinity media
Preparation of DNA affinity media
This experiment describes the preparation process of DNA affinity medium. This experiment is from the Laboratory Guide for Protein Purification and Characterization, by Zhu Houzhu.
Operation method
Preparation of DNA affinity media
Materials and Instruments
ATP [y-32P]ATP T4 polynucleotide kinase Ammonium acetate Ethanol Phenol Chloroform Chloroform Isoamyl Alcohol NaOAc T4 DNA ligase Phenol Isopropyl Alcohol Re-distilled water Sepharose CL-2B Cyanogen bromide N N-Dimethylformamide NaOH Potassium phosphate KCl CNBr-Activated Sepharosundefined.4B HCl T4 polynucleotide kinase Buffer TE Junction-Kinase Buffer Ethanolamine-HCl Storage Buffer for Chromatography Columns Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SpeedVec Rotary Concentrator
ATP (20 mmol/L; pH 7.0)
[ y-32P ]ATP (5uCi)
T4 polynucleotide kinase (10 U/ul) (Stratagene)
Ammonium acetate (10mol/L)
Ethanol (100% and 75%; v/v)
Phenol/chloroform (1:1;v/v)
Chloroform/Isoamyl alcohol (3-methyl-1-butanol) (24:1;v/v)
NaOAc (3mol/L)
T4 DNA ligase (Stratagene)
Phenol (equilibrated with TE)
Isopropanol (2-propanol)
Redistilled water
Sepharose CL-2B (pharmacia Biotech, Inc.)
Cyanogen bromide (CNBr)(Aldrich Chemical Co., C9149-2)
N,N-Dimethylformamide
NaOH(5mol/L)
Potassium phosphate (lOmmol/L and lmol/Lt both pH 8.0)
KCl(1mol/L)
CNBr-activated Sepharosundefined.4B (Pharmacia Biotech, Inc.)
HCl(1 mmol/L)
SpeedVec Rotary Concentrator (Savant Instruments, Inc.)
Reagents
T4 polynucleotide kinase buffer (10X)
TE
Junction-kinase buffer (10x)
Ethanolamine-HCl (1 mol/L; pH 8.0) [1 mol/L ethanolamine (free base) was adjusted to pH 8.0 with HC1].
Storage buffer for chromatography columns
(For recipe, see "Preparation of Reagents", P.131-138)
Operating Procedures
5'-phosphorylation of oligonucleotides
1) Prepare the following reaction mixture:
DNA (440ug per oligonucleotide dissolved in TE; i.e., total DNA 880ug) 65ul
T4 Polynucleotide Kinase Buffer (10X) 10ul
Total volume: 75ul
2) Denaturation and annealing of complementary DNA double strand: Incubate at 88°C for 2 min, 65°C for 10 min, 37°C for 10 min, and finally incubate at room temperature for 5 min.
3) Combine the following solutions:
DNA (obtained from the previous step) 75ul
ATP (20 mml/L; pH 7.0; containing ~5uCi[ γ-32P ]ATP) 15ul
T4 polynucleotide kinase (100U) 10ul
Total volume: 100ul
4) Incubate at 37°C for 2 h, add 50ul of 10mol/L ammonium acetate and 100ul of H20 to make a final volume of 250ul.
5) Heat at 65°C for 15 min to inactivate the kinase.
6) Bring the mixture to room temperature. Add 750ul of 100% ethanol and mix well by inverting. Centrifuge the mixture at high speed for 15 min at room temperature to precipitate the DNA.
7) Discard the supernatant, add 225ul of TE, shake, and dissolve the precipitate. c
8) Add 250u1 of 1:1 phenol/chloroform, shake and mix for 1 min, and centrifuge at high speed for 5 min to separate the two phases.
9) Transfer the upper liquid layer to a new tube, add 250ul 24:1 chloroform/isoamyl alcohol, shake and mix for 1 min, centrifuge at high speed for 5 min to separate the two phases.
10) Transfer the upper layer to a new tube, add 25ul 3mol/L NaOAc, shake and mix.
11) Add 750ul of 100% ethanol, mix upside down and centrifuge at high speed for 15 min to precipitate the DNA.
12) Discard the supernatant, add 800ul of 75% ethanol, mix with shaking and centrifuge at high speed for 5 min.
13) Discard the supernatant and dry in SpeedVac rotary concentrator.
Ligation of Oligonucleotides
1) Take 10ul of 10X Junction-Kinase Buffer and add it to 65ul of water. Shock dissolve the DNA in this solution.
2) Add 20ul of 20 mmol/L ATP (pH 7.0) and 5ul of T4 DNA Ligase (30 Weiss units) to give a final reaction volume of 100ul.
3) Incubate at room temperature for ≥2 h. If the ligation of AP-1 oligonucleotides does not proceed, try raising the temperature to 30°C.
Note: The optimal temperature for the ligation reaction can vary from 4°C to 30°C depending on the cold oligonucleotide used. Short oligonucleotides (≤pentadecamer) are better ligated at lower temperatures (4~15℃), while oligonucleotides with medium degree of palindromic structure have a tendency to anneal by themselves, so it is better to ligate them at higher temperatures (15-30℃).
(4) Monitor the progress of the ligation reaction by agarose gel electrophoresis (0.5ul of ligated reactants for each lane, the average length of the ligated oligonucleotides is usually at least an undecamer.
Note: 1) 5'-phosphorylated oligonucleotides often fail to ligate at the beginning. Therefore, if the ligation reaction does not occur, the DNA can be extracted once with 1:1 phenol/chloroform and again with chloroform, followed by ethanol precipitation (with NaOAc as salt). Dissolve DNA in 225ul TE, add 25ul of 3mol/L NaOAc, reprecipitate with 750ul of 100% ethanol n Precipitates are washed with 75% ethanol and dried in a SpeedVac concentrator before attempting ligation. n If the DNA is not in the ligand, the ligand should be extracted once with 1:1 phenol/chloroform, then once with chloroform. ② If possible, oligonucleotides should be ligated to at least decamer length. However, if effective ligation cannot be achieved, it is possible to prepare DNA affinity media from poorly ligated DNA fragments, since in many cases it has been found that affinity chromatography can still be performed efficiently with monomers of complementary oligonucleotides.
Preparation of DNA for coupled Sepharose
Remove residual ATP by ammonium acetate-isopropanol precipitation, otherwise it will interfere with the coupling of the ligated DNA to CNBr-activated Sepharose.
1) Add 100ul of phenol (already equilibrated with TE) to 100-ul of ligated reaction, shake and mix for 1 min. centrifuge for 5 min at room temperature, and transfer the upper layer to a new tube.
2) Add 100ul of 24:1 phenol/isoamyl alcohol, shake and mix for 1 min; centrifuge at high speed in a microcentrifuge for 5 min at room temperature; transfer the upper layer to a new tube.
3) Add 33ul 10mol/L ammonium acetate and mix with shaking.
4) Add 133ul isopropanol (2-propanol), mix upside down; incubate at -20℃ for 20 min; centrifuge at high speed for 15 min to precipitate DNA; discard supernatant.
5) Add 225ul of TE, shake to dissolve the precipitate, add 25ul of 3mol/L NaOAc, shake to mix; add 750ul of 100% ethanol, invert and mix well; centrifuge at high speed for 15 min to precipitate DNA; discard the supernatant.
6) Wash the resulting DNA with 75% ethanol for two times and dry the precipitate in SpeedVac concentrator.
7) Dissolve DNA in 50 ml of redistilled water and store at -20℃. Do not dissolve DNA in TE, the Tris buffer in TE will interfere with the coupling reaction.
Couple DNA with Sepharose using cyanogen bromide.
This procedure consists of activating Sepharose CL-2B with cyanogen bromide (CNBr), and then coupling the ligated DNA to the CNBr-activated Sepharose. Since CNBr is highly toxic, many researchers prefer the alternative method described in the following section, i.e., to use commercially available CNBr-activated Sepharose, which does not involve direct manipulation with CNBr. Activation with CNBr provides flexibility in the choice of medium. We generally choose Sepharose CL-2B, which is a cross-linked form of Sepharose 2B with higher strength. However, the only commercially available CNBr-activated medium is Sepharose 4B. Another advantage of using CNBr directly is that the cost of preparing the activation medium is lower than purchasing pre-activated medium. Finally, although the DNA affinity media prepared by both methods may be equally effective in purifying specific sequences of DNA-binding proteins, the procedures described in this section are more widely used than those given in the following section.
1) In a 60-ml coarse sintered slide funnel, wash 10 to 15 ml (of the deposited bed SepharoseCL-2B thoroughly with redistilled water. about 500 ml of water is used to wash the gel.
2) Transfer the wet Sepharose gel to a graduated 25-ml measuring cylinder, measure about 10 ml of the deposited gel, and add water to a final volume of 20 ml. Transfer this slurry to a 150-ml glass boiler placed in a water bath (equilibrated to 15°C), which is placed on a magnetic mixer in a fume hood.
3) In the fume hood, weigh 1.lg of CNBr into a 25-ml conical flask and seal with paraffin film. It is desirable that the amount of CNBr be slightly more than, but not slightly less than, 1.1 g. Dissolve CNBr in 2 ml of N,N-dimethylformamide; CNBr will dissolve immediately. Add the CNBr solution dropwise to the slurry Sepharose gel over a period of 1 minute while stirring.
4) Immediately add NaOH as described below. add 30ul of 5mol/L NaOH to the above mixture every 10s with stirring (at 15°C) for 10 min, until the total volume of NaOH added is 1.8 ml.
5) Immediately add 100 ml of ice-cold water to the beaker and pour the mixture into a 60-ml coarse sintered glass funnel. At this point, it is extremely important not to pipette the gel into a dry cake. In the event that the gel is inadvertently strained to a dry cake during aspiration, do not use this dry gel again and start again from step 1).
6) Wash the gel 4 times with 100 ml of ice-cold water (≤4°C), and then wash it twice with 100 ml of ice-cold 10mol/L potassium phosphate (PH8.0).
7) Immediately transfer half of the gel (5 ml) into a screw-capped 15-ml polypropylene tube and add about 2 ml of 10 mmol/L potassium phosphate (pH 8.0) until the gel forms a thick paste.
8) Immediately add DNA (dissolved in 50ul of water, totaling 880ug). Incubate overnight at room temperature on a turntable.
9) Transfer the gel to a 60-ml coarse sintered slide funnel, wash twice with 100 ml of water, and then wash once with 100 ml lmol/L ethanolamine-HC1 (pH 8.0).
Note; Compare the level of radioactivity in the first few milliliters of filtrate with that in the medium after washing to estimate the efficiency of DNA binding to the medium. General. All detectable radioactivity is present only in the medium.
10) Transfer the gel to a 5-ml polypropylene tube with a screw cap and add 1 mol/L ethanolamine-HCl (pH 8.O) until the mixture is a smooth slurry. The purpose of this step is to inactivate the unreacted CNBr-activated Sepharose.
11) Wash the gel with the following solution:
10 mmol/L potassium phosphate buffer (pH 8.0) 100 ml
1 mol/L potassium phosphate buffer (pH 8.0) 100 ml
1mol/L KCl 100 ml
H20 100 ml
Column preservation buffer 100 ml
12) Store the prepared gel at 4°C (do not freeze). The gel is stable for at least 1 year.
Coupling of DNA to Sepharose using CNBr-activated Sepharose This operation, starting with commercially available CNBr-activated Sepharose, is safer and easier than the method described in the previous section, which involves the preparation of CNBr-activated media.
1) Weigh 2 g of CNBr-activated Sepharose4B gel.
Note: 1 g of frozen material yields a final volume of approximately 3.5 ml.
2) In a sintered glass funnel, wash and swell the gel with 400 ml of lmml/LHC1 for about 15 min.
3) Wash the gel with 100 ml of H2O, then with 100 ml of 10 mmol/L potassium phosphate (pH 8.0).
4) Immediately transfer 5 ml of gel to a 15-ml polypropylene tube with a screw cap and add about 2 ml of 10 mmol/L potassium phosphate (pH 8.0) until a viscous slurry is formed.
5) Immediately add DNA (dissolved in 50ul H2O, totaling 880ul). Incubate at room temperature on a rotor for 5~6 h (overnight if needed).
6) Transfer the gel to a 60-ml coarse sintered slide funnel and wash twice with 100 ml H20 and once with 100 ml 1mol/L ethanolamine-HC1 (pH 8.0).
Note: Compare the level of radioactivity in the first few milliliters of filtrate with that in the medium after washing to estimate the efficiency of DNA binding to the medium. Typically, all detectable radioactivity is present only in the medium a
7) Transfer the gel to a screw-cap 15-ml polypropylene tube and add lmol/L ethanolamine-HCl (pH 8.0), until the mixture is a smooth slurry. b) Add the gel to a 15-ml polypropylene tube with a screw cap and add lmol/L ethanolamine-HCl (pH 8.0), until the mixture is a smooth slurry. Incubate on a rotor at room temperature for 4-6 h. The purpose of this step is to inactivate the unreacted CNBr-activated Sepharose.
8) Wash the gel with the following solution:
10 mmol/L potassium phosphate buffer (pH 8.0) 100 ml
1 mol/L potassium phosphate buffer (pH 8.0) 100 ml
1mol/L KCl 100 ml
H20 100 ml
Column preservation buffer 100 ml
9) Store the prepared gel at 4°C (do not freeze). The gel is stable for at least 1 year.