AGS or AGSΔUNG cells were transfected with 1 μg BKRF3 or vector control using 3 μL TransIT-LT1 in serum free RPMI. Cells were harvested after 30 hrs and were lysed in 300 μL modified HED buffer (20 mM HEPES, 15 mM EDTA, Roche cOmplete EDTA-free protease inhibitor cocktail tablet, pH 7.4) per 106 cells. Activity assays were carried out as described12 (link) using a 10 minute incubation with a dU-containing oligo (RSH12955 5’-AAA AAA AAA UCG GGA AAA AAA-fluorescein-3’). 2 μL of UGI (4 units, NEB M0281S) were used to inhibit UDG. Products were separated by a 20% TBE-Urea PAGE. Separated DNA fragments were visualized on a Typhoon FLA-7000 scanner on fluorescence mode.
Protocol detail
BKRF3 Protein Activity Assay Protocol
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Assays
Buffer
Cells
Edta
Fluorescein
Fluorescence
Hepes
Inhibit
Oligo
Protease inhibitor
Serum
Tablet
Typhoon
Urea
Corresponding Organization :
Other organizations : University of Minnesota, University of Toronto, Ontario Institute for Cancer Research, University of Minnesota Medical Center
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Variable analysis
independent variables
- BKRF3 or vector control
- DNA fragments visualized on a Typhoon FLA-7000 scanner on fluorescence mode
- AGS or AGSΔUNG cells
- 1 μg BKRF3 or vector control
- 3 μL TransIT-LT1 in serum free RPMI
- Harvested after 30 hrs
- Lysed in 300 μL modified HED buffer (20 mM HEPES, 15 mM EDTA, Roche cOmplete EDTA-free protease inhibitor cocktail tablet, pH 7.4) per 10^6 cells
- 10 minute incubation with a dU-containing oligo (RSH12955 5'-AAA AAA AAA UCG GGA AAA AAA-fluorescein-3')
- 2 μL of UGI (4 units, NEB M0281S) used to inhibit UDG
- 20% TBE-Urea PAGE used to separate products
- vector control
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