Experiment Protocol 301

Immobilization of double strand DNA on Plain beads
This experiment protocol describes the immobilization of double strand DNA to purify DNA-bound proteins such astranscription factors. This protocol includes annealing of complementary single strand DNA, 5’ end phosphorylation, ligation,and immobilization on beads. Double strand DNA is immobilized on beads by reacting guanine amino groups of DNA
protruding ends with epoxy groups on beads surfaces.

1. Materials
1.1 Beads and Ligands (DNA)・Plain beads (TAS8848N1010): 20mg (Functional groups: Approx. 1µmol/mg)
・Complementary single strand DNA: approximately 150µg for each annealing
1. Target sequence (Example)
5’-AGGGTATGCAAATTAAGAAG-3’
3’-TCCCATACGTTTAATTCTTC-5’
2. Synthetic oligonucleotide for immobilization
Sense:
5’-GGGGAGGGTATGCAAATTAAGAAG-3’
Antisense: 3’-TCCCATACGTTTAATTCTTCCCCC-5’
1.2 Reagents
・T4 Polynucleotide Kinase (Takara Bio 2021S, etc.)
・T4 DNA Ligase (Takara Bio 2011A, etc.)
・Phenol/Chloroform 2mL ・Sodium chloride ・Ethanol 10mL
・NICK columns (GE Healthcare 17-0855-02, etc.)
・3M Sodium acetate (pH5.3) ・2.5M Potassium chloride ・Agarose gel
・TES buffer
10 mM Tris-HCl (pH 8.0)
0.3 M KCl
1 mM EDTA
0.02 % NaN3
1.3 Apparatus
・Micro high-speed cold centrifuge ・Thermostat bath ・Electrophoresis unit
・Spectrophotometer ・UV lamp or imager ・Vortex mixer
・Ultrasonic dispersing device
We have performed operation checks with an ultrasonic homogenizer: VP-15S with a cuphorn(TAITEC), and an ultrasonic dispersing device: TA4905 (Tamagawa Seiki).
2. Method
2.1 Outline
The following is a schematic view of ligand immobilization. Refer to the next section 2.2“Procedures” for details.

Annealing

Phosphorylation of 5´terminus

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Experiment Protocol 301

Ligation

Plain beads

DNA immobilized beads

2.2 Procedures
2.2.1 Annealing
1) Dilute each single strand DNA with ultrapure water to adjust its concentration to 1µg/µL.
2) Add 150µg of sense oligonucleotide and 150µg of antisense oligonucleotide into a 1.5mL
micro-tube as below, and mix them.

3)

Sense oligonucleotide

150µL(150µg)

Antisense oligonucleotide

150µL(150µg)300µL(300µg)Heat the solution for ten minutes at 98℃, float it in 200mL of water at 98℃, and leave it
until it cools down to room temperature (overnight).

2.2.2 PNK (polynucleotide kinase) treatment of DNA ends
Note: When using 5’ end phosphorylated synthetic oligonucleotide, skip this process.
1) Divide the annealed DNA into eight tubes, mix each of them with PNK as below, and standthem still for 60 minutes at 37℃ to react them.
DNA35µL

10× Kinase buffer

10µL

10 mM ATP10µL

H2O

43.2µLT4 Polynucleotide Kinase

1.8µL(35µg)

100µL2)

Add 100µL of phenol/chloroform, and mix them for 30 seconds. (Vortex mixer)

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Experiment Protocol 301
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)

Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer a water layer
to a fresh 1.5mL micro-tube.
Add 10µL of 3M sodium acetate (pH5.3) and 250µL of ethanol, and mix them.
Stand the solution still for one hour at －30℃ to precipitate the ethanol.
Centrifuge at 15,000 rpm for 30 minutes at 4℃, and discard the supernatant.
Add 500µL of 70% ethanol gently, and rinse them.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Dry them. (Vacuum drying, etc.)
Add 10µL of ultrapure water, and dissolve them.
Collect the DNA solutions into one tube. (80µL in total)
Quantify the volume of DNA, and adjust its concentration to 1µg/µL. (280µg /280µL)

2.2.3 Ligation
1) Divide the annealed DNA into eight tubes, and mix each of them with DNA Ligase as below.
DNA25µL

10× Ligation buffer

10µL

H2O

63µL

T4 DNA ligase

2µL(25µg)

100µL2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)

After spin down, stand them still at 4℃ to react them overnight.
Measure the length of DNA with agarose gel. (Target length：200 to 400bp)。
Add 100µL of ultrapure water. (Fluid volume: 200µL)
Add 200µL of phenol/chloroform, and mix them for 30 seconds. (Vortex mixer)
Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer a water layer toa fresh 1.5mL micro-tube.
Add 20µL of 3M sodium acetate (pH5.3) and 500µL of ethanol, and mix them.
Stand the solution still for one hour at －30℃ to precipitate the ethanol.
Centrifuge at 15,000 rpm for 30 minutes at 4℃, and discard the supernatant.
Add 1mL of 70% ethanol gently, and rinse them.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Dry them. (Vacuum drying, etc.)
Add 10µL of ultrapure water, and dissolve them.
Collect the eight DNA solutions into one tube. (80µL in total)
Load the solution onto NICK columns. (Equilibrate the NICK columns with ultrapure waterprior to loading.)
Elute them four times with 400µL of ultrapure water.
Perform quantification of nucleic acids on each recovered fraction, and store a sample (2fractions) which has the highest concentration. (The DNA concentration: approximately
250µg/mL)

2.2.4 Immobilization of double strand DNA on beads
1) Add 10mg of Plain beads into each of two 1.5 mL micro-tubes.
2) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
3) Add 500µL of ultrapure water, and disperse the beads.
4) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.

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Experiment Protocol 301
5)6)

Repeat the above 3) to 4) two more times. (Wash the beads twice in total.)
Add 400µL of ultrapure water into one of the two micro-tubes to control the solution, and add
400µL of the ligated DNA solution (250µg/mL) into the other micro-tube as below, anddisperse the beads. (When you cannot disperse the beads easily, disperse them in a shorttime by using an ice-cold ultrasonic homogenizer.)

DNAH2O－0µL
400µL＋400µL
0µL(100µg)7)
8)Stand the solutions still for 24 hours at 50℃ to react them.
Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer thesupernatant to another tube.
9) Add 400µL of 2.5M potassium chloride, and disperse the beads.
10)
Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer thesupernatant to another tube.
12) Repeat the above 9) to 10) one more time. (Wash the beads twice in total.)13) Add 400µL of ultrapure water, and disperse the beads.
14)Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer thesupernatant to another tube.
15) Repeat the above 13) to 14) one more time. (Wash the beads twice in total.)16) Add 400µL of TES buffer, and disperse the beads.
17) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
18) Repeat the above 16) to 17) two more times. (Wash the beads three times in total.)19) Disperse them into 400µL of TES buffer, and store them at 4℃. (The concentration of double
strand DNA immobilized beads：25mg/mL)
Note: The volume of DNA immobilization is approximately 2µg/mg beads (10pmol/mg).
(Calculate the volume by subtracting the volume of DNA in the washing supernatant and thevolume of DNA in the reaction supernatant from the volume of the input DNA.)
3. Supplements
・ Beads are easily dispersed by using an ultrasonic dispersing device. But if you do not have such adevice, they are dispersed by using an ultrasonic washer, or by the manual agitation. In the manual
dispersion method, the bottom of a micro-tube is glided over an uneven surface (side of plastic test
tube rack in this case) creating turbulence through the collisions. (see left side picture below)
Please make sure to use well-constructed tubes with the caps tightly secured in order to prevent
leakage/breakage. Use of cap lock is recommended in order to prevent leakage. (see right side
picture below).
For more information, please visit FG beads web site and see the movie of the method.
(Please click : http://www.magneticnanoparticle.jp/en/htdocs/technique/affinity.html for movingpictures.)

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