AluI R6281

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	DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. DNA digestion was performed on cells in three-dimensional cultures grown for 14 days.Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period.

Protocol tips

DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. DNA digestion was performed on cells in three-dimensional cultures grown for 14 days.Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period.

Publication protocol

"Cell smear assays were performed as described previously.1 In brief, monolayer cultures grown on 10-cm dishes (70 to 90% confluent) were mechanically dislodged or trypsinized off the plate, collected by centrifugation, and resuspended in serum-free DMEM. A drop (20 μl) of the suspension was placed on a glass slide and dried for 1 hour. DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. Nuclei were observed and photographed with a Leica inverted fluorescent microscope (Leica, Bannockburn, IL).

DNA digestion was performed on cells in three-dimensional cultures grown for 14 days. In preliminary experiments, we first determined both the time and concentration of AluI required for DNA digestion, taking into account that large aggregates of T4-2 cells contain more DNA than smaller MCF10A acini. Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures.

Both Triton X-100 (0.1%) and Nonidet P-40 (0.1%) were tested as detergents topermeabilize cells in three-dimensional cultures to explore if the detergent per se affected DNA digestion. No difference in sensitivity to DNA digestion was observed using either one of these detergents; however, when compared with cells treated with Triton X-100, the morphology of cells permeabilized with Nonidet P-40 was better restored to a round shape, making it easier to evaluate the results. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period. Ethidium bromide (0.5 μg/ml) was added to label DNA at the termination of the reaction. Nuclear fluorescence was photographed with a Leica inverted fluorescent microscope. Parallel cultures in each experiment were stained with trypan blue (MP Biomedicals, Aurora, OH) to confirm complete permeabilization"

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