2. Heparin solution stock (10 mg/mL) is diluted in ultrapure
RNase-free water.
3. 10Â Running Buffer (100 mM potassium phosphate, pH 8.0,
800 mM NaCl, 60 mM MgCl 2 , 0.5% Tween 20).
4. Amicon Ultra-4 centrifugation filter unit 30,000 MWCO
(Millipore, Billerica, MA, USA).
3 Methods
3.1 Polymerase
Preparation
for switchSENSE
Experiment
1. Aliquots of HIV-1 reverse transcriptase (ammonium sulfate
stock solution) are centrifuged for 20 min at 15,000 Â g,
4
C. Protein pellets are resuspended for 1 h on ice in the 1Â
Running Buffer at final concentration of ~1 mg/mL.
2. The protein is then dialyzed against 1Â Running Buffer using
an Amicon Ultra-4 by three successive 10-min centrifugations
at 7,500 Â g, 4
C.
3. After the last centrifugation step, the polymerase is concentrated at a final concentration of ~1 mg/mL.
4. Precise concentration of polymerase is obtained using two
independent measures with a nanodrop spectrophotometer
(ε280 ¼ 231,480 mol
À1 cm
À1 for pure HIV-1 reverse
transcriptase).
3.2 Solutions
Preparation
1. 300 μL of RT diluted with 1Â Running Buffer (ranging from
0.03 to 4 nM, based on concentration estimated in the
previous step).
2. 250 mL of freshly diluted 1Â Auxiliary Buffer and 1Â Running
Buffer are filtrated on a 2-μm nitrocellulose filter, inserted in
the fluidic system of the DRX 2400 and primed using the
switchCONTROL command of the system software.
3. 300 μL of p36T80 oligonucleotide diluted in ultrapure water
at a final concentration of 500 nM.
4. 1 mL of regeneration solution.
5. 10 mL of freshly diluted 1Â passivation solution.
6. All the solutions and buffers are arranged as shown in the
switch CONTROL software window.
3.3 Schematic
Overview
of the Experiment
1. Building of the DNA nanolever is the first step of the experiment. This reaction is achieved by the hybridization of the
P36T80 oligonucleotide on the 80-mer DNA probe attached
to the chip (3
0 end), to generate a DNA duplex with a 44 bases
5
0 overhang (Fig. 1a, see Note 2).
148
Guillaume Bec and Eric Ennifar
RNase-free water.
3. 10Â Running Buffer (100 mM potassium phosphate, pH 8.0,
800 mM NaCl, 60 mM MgCl 2 , 0.5% Tween 20).
4. Amicon Ultra-4 centrifugation filter unit 30,000 MWCO
(Millipore, Billerica, MA, USA).
3 Methods
3.1 Polymerase
Preparation
for switchSENSE
Experiment
1. Aliquots of HIV-1 reverse transcriptase (ammonium sulfate
stock solution) are centrifuged for 20 min at 15,000 Â g,
4
C. Protein pellets are resuspended for 1 h on ice in the 1Â
Running Buffer at final concentration of ~1 mg/mL.
2. The protein is then dialyzed against 1Â Running Buffer using
an Amicon Ultra-4 by three successive 10-min centrifugations
at 7,500 Â g, 4
C.
3. After the last centrifugation step, the polymerase is concentrated at a final concentration of ~1 mg/mL.
4. Precise concentration of polymerase is obtained using two
independent measures with a nanodrop spectrophotometer
(ε280 ¼ 231,480 mol
À1 cm
À1 for pure HIV-1 reverse
transcriptase).
3.2 Solutions
Preparation
1. 300 μL of RT diluted with 1Â Running Buffer (ranging from
0.03 to 4 nM, based on concentration estimated in the
previous step).
2. 250 mL of freshly diluted 1Â Auxiliary Buffer and 1Â Running
Buffer are filtrated on a 2-μm nitrocellulose filter, inserted in
the fluidic system of the DRX 2400 and primed using the
switchCONTROL command of the system software.
3. 300 μL of p36T80 oligonucleotide diluted in ultrapure water
at a final concentration of 500 nM.
4. 1 mL of regeneration solution.
5. 10 mL of freshly diluted 1Â passivation solution.
6. All the solutions and buffers are arranged as shown in the
switch CONTROL software window.
3.3 Schematic
Overview
of the Experiment
1. Building of the DNA nanolever is the first step of the experiment. This reaction is achieved by the hybridization of the
P36T80 oligonucleotide on the 80-mer DNA probe attached
to the chip (3
0 end), to generate a DNA duplex with a 44 bases
5
0 overhang (Fig. 1a, see Note 2).
148
Guillaume Bec and Eric Ennifar