9. Determine the total protein content in each fraction using the
sample volume, and calculate the recovery in % by comparing
the total protein amount in the individual fractions with the
total protein amount in the feed solution.
3.2.8 Determination
of the Total dsDNA Amount
1. Prepare 1Â TE-buffer in a 50-mL reaction tube, by diluting the
kit-contained 20Â buffer with deionized water. For the above
performed chromatographic run with four samples, about
12 mL should be prepared.
2. Prepare the standard calibration samples using the lambdaDNA stock solution contained in the kit, as described in the
instructions.
3. Dilute the Quant-iT™ PicoGreen
® dsDNA reagent 1:200 with
1Â TE-buffer in a fresh 50-mL reaction tube. Prepare enough
reagent to apply 100 μL of the mix per well, and consume
within 1 h.
4. Pipet 100 μL of each standard and blank (duplicates) into a
black 96-well microplate.
5. Transfer 20 μL of each chromatographic sample in duplicates
(see Note 54).
6. Add 80 μL 1Â TE buffer to each 20 μL sample.
7. Add 100 μL working reagent to all wells (standards, blanks and
samples).
8. Shake the plate gently and let it incubate in the dark for 5 min.
9. Measure the emission at 520 nm after fluorescence excitation at
480 nm in a plate reader.
10. Prepare the standard calibration curve and calculate DNA concentrations and recoveries as described in Subheading 3.2.7 for
the total protein amount (see Note 55).
4 Notes
Upstream processing
Thawing cells
1. Use up to 10% FBS (Biochrom, Berlin, Germany).
2. Ensure the thread of the cryovial is above the warm water when
thawing the cells to avoid a contamination risk.
Cell passaging
3. Apply the shake-off technique, if cells don’t detach. Use the
heel of your hand to gently knock via the side of the T-flask,
until the cells detach. Check the detachment under the light
microscope.
238
Keven Lothert et al.
sample volume, and calculate the recovery in % by comparing
the total protein amount in the individual fractions with the
total protein amount in the feed solution.
3.2.8 Determination
of the Total dsDNA Amount
1. Prepare 1Â TE-buffer in a 50-mL reaction tube, by diluting the
kit-contained 20Â buffer with deionized water. For the above
performed chromatographic run with four samples, about
12 mL should be prepared.
2. Prepare the standard calibration samples using the lambdaDNA stock solution contained in the kit, as described in the
instructions.
3. Dilute the Quant-iT™ PicoGreen
® dsDNA reagent 1:200 with
1Â TE-buffer in a fresh 50-mL reaction tube. Prepare enough
reagent to apply 100 μL of the mix per well, and consume
within 1 h.
4. Pipet 100 μL of each standard and blank (duplicates) into a
black 96-well microplate.
5. Transfer 20 μL of each chromatographic sample in duplicates
(see Note 54).
6. Add 80 μL 1Â TE buffer to each 20 μL sample.
7. Add 100 μL working reagent to all wells (standards, blanks and
samples).
8. Shake the plate gently and let it incubate in the dark for 5 min.
9. Measure the emission at 520 nm after fluorescence excitation at
480 nm in a plate reader.
10. Prepare the standard calibration curve and calculate DNA concentrations and recoveries as described in Subheading 3.2.7 for
the total protein amount (see Note 55).
4 Notes
Upstream processing
Thawing cells
1. Use up to 10% FBS (Biochrom, Berlin, Germany).
2. Ensure the thread of the cryovial is above the warm water when
thawing the cells to avoid a contamination risk.
Cell passaging
3. Apply the shake-off technique, if cells don’t detach. Use the
heel of your hand to gently knock via the side of the T-flask,
until the cells detach. Check the detachment under the light
microscope.
238
Keven Lothert et al.