6. Pool fractions containing high amounts of template/primer
and store aliquots of 100 μL at À20
C until use. The concentration of the template/primer solution is typically about
2.5 μM.
7. Verify the homogeneity of the template/primer by AUC sedimentation velocity experiments in buffer C at 50,000 rpm
using the absorbance optics at 260 nm (as shown in Fig. 4d)
or by gel electrophoresis followed by staining with SYBR Gold
(as shown in Fig. 4e).
3.3 Sample
Preparation
1. Centrifuge protein solutions after thawing for 20 min at
15,500 Â g and 4
C to remove aggregates, which would
interfere with the correct determination of protein concentration. Add an appropriate volume of buffer A or buffer A
supplemented with 1 μM BSA, respectively, to achieve a concentration of about 15 μM SSB and 1 μM FITC-labeled clamp
loader. Thaw 55-mer/30-mer primer/template solution.
2. Measure absorbance spectra of unlabeled protein and DNA
samples in the wavelength range of 320 to 220 nm (see Note
4) or 600 to 220 nm in the case of FITC-labeled clamp loader.
Calculate protein and DNA concentrations using the extinction coefficients given above.
3. Prepare template/primer-SSB solution by mixing of 2.2 μM
55-mer/30-mer template/primer with 2.5 μM SSB (1.1-fold
excess of SSB over template/primer). As template/primer is
stored in a buffer without TCEP and MgCl 2 , appropriate
volumes of TCEP and MgCl 2 stock solutions have to be
added in order to adjust for composition of buffer A.
4. Prepare 9–14 AUC samples with a total volume of 110 μL by
mixing the components in the following order: an appropriate
volume of buffer A, BSA to a final concentration of 1 μM,
2 mM ATP, 55-mer/30-mer template/primer-SSB in increasing concentrations, and 100 nM FITC-labeled clamp loader. In
the experiment shown in Fig. 5, 55-mer/30-mer template/
primer was added in the following concentrations: 0, 50 nM,
100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 1.5 μM, and 2 μM.
3.4 Performing
the Analytical
Ultracentrifugation
Experiments
1. Assemble seven analytical ultracentrifugation cells using standard 3-mm two-channel charcoal-filled Epon centerpieces and
special fluorescence cell housings (Fig. 1c) according to the
Beckman Coulter rotor manual LXL/A-TB-003F provided
with the centrifuge (see Note 5).
2. Fill each channel with 100 μL of sample (see Note 6). No
reference is needed in fluorescence measurements; therefore,
both channels can be loaded with samples (see Note 7).
412
Andrea Bogutzki and Ute Curth
and store aliquots of 100 μL at À20
C until use. The concentration of the template/primer solution is typically about
2.5 μM.
7. Verify the homogeneity of the template/primer by AUC sedimentation velocity experiments in buffer C at 50,000 rpm
using the absorbance optics at 260 nm (as shown in Fig. 4d)
or by gel electrophoresis followed by staining with SYBR Gold
(as shown in Fig. 4e).
3.3 Sample
Preparation
1. Centrifuge protein solutions after thawing for 20 min at
15,500 Â g and 4
C to remove aggregates, which would
interfere with the correct determination of protein concentration. Add an appropriate volume of buffer A or buffer A
supplemented with 1 μM BSA, respectively, to achieve a concentration of about 15 μM SSB and 1 μM FITC-labeled clamp
loader. Thaw 55-mer/30-mer primer/template solution.
2. Measure absorbance spectra of unlabeled protein and DNA
samples in the wavelength range of 320 to 220 nm (see Note
4) or 600 to 220 nm in the case of FITC-labeled clamp loader.
Calculate protein and DNA concentrations using the extinction coefficients given above.
3. Prepare template/primer-SSB solution by mixing of 2.2 μM
55-mer/30-mer template/primer with 2.5 μM SSB (1.1-fold
excess of SSB over template/primer). As template/primer is
stored in a buffer without TCEP and MgCl 2 , appropriate
volumes of TCEP and MgCl 2 stock solutions have to be
added in order to adjust for composition of buffer A.
4. Prepare 9–14 AUC samples with a total volume of 110 μL by
mixing the components in the following order: an appropriate
volume of buffer A, BSA to a final concentration of 1 μM,
2 mM ATP, 55-mer/30-mer template/primer-SSB in increasing concentrations, and 100 nM FITC-labeled clamp loader. In
the experiment shown in Fig. 5, 55-mer/30-mer template/
primer was added in the following concentrations: 0, 50 nM,
100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 1.5 μM, and 2 μM.
3.4 Performing
the Analytical
Ultracentrifugation
Experiments
1. Assemble seven analytical ultracentrifugation cells using standard 3-mm two-channel charcoal-filled Epon centerpieces and
special fluorescence cell housings (Fig. 1c) according to the
Beckman Coulter rotor manual LXL/A-TB-003F provided
with the centrifuge (see Note 5).
2. Fill each channel with 100 μL of sample (see Note 6). No
reference is needed in fluorescence measurements; therefore,
both channels can be loaded with samples (see Note 7).
412
Andrea Bogutzki and Ute Curth