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E8263 25ku

Manufactured by Merck Group

The E8263-25KU is a laboratory equipment product manufactured by Merck Group. It serves a core function as a laboratory instrument, but a detailed description while maintaining an unbiased and factual approach is not available.

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2 protocols using e8263 25ku

1

Enhancer AAV Vector Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
Enhancer AAV plasmids were maxi-prepped and transfected with PEI Max 40K (Polysciences Inc., catalog # 24765-1) into one 15 cm plate of AAV-293 cells (Cell Biolabs catalog # AAV-100), along with helper plasmid pHelper (Cell BioLabs) and PHP.eB rep/cap packaging plasmid (Chan et al., 2017 (link)), with a total mass of 150 μg PEI Max 40K, 30 μg pHelper, 15 μg rep/cap plasmid, and 15 μg enhancer-AAV vector. The next day medium was changed to 1% FBS, and then after 5 days cells and supernatant were harvested and AAV particles released by three freeze-thaw cycles. Lysate was then treated with benzonase to degrade free DNA (2 μL benzonase, 30 min at 37°C, MilliporeSigma catalog # E8263-25KU), and then cell debris was cleared with low-speed spin (1500 g 10 min). The supernatant containing virus was concentrated over a 100 kDa molecular weight cutoff Centricon column (MilliporeSigma catalog # Z648043) to a final volume of ~150 μL. For highly purified large-scale preps this protocol was altered so that ten plates were transfected and harvested together at 3 days after transfection, and then the crude virus was purified by iodixanol gradient centrifugation.
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2

Enhancer AAV Vector Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
Enhancer AAV plasmids were maxi-prepped and transfected with PEI Max 40K (Polysciences Inc., catalog # 24765-1) into one 15 cm plate of AAV-293 cells (Cell Biolabs catalog # AAV-100), along with helper plasmid pHelper (Cell BioLabs) and PHP.eB rep/cap packaging plasmid (Chan et al., 2017 (link)), with a total mass of 150 μg PEI Max 40K, 30 μg pHelper, 15 μg rep/cap plasmid, and 15 μg enhancer-AAV vector. The next day medium was changed to 1% FBS, and then after 5 days cells and supernatant were harvested and AAV particles released by three freeze-thaw cycles. Lysate was then treated with benzonase to degrade free DNA (2 μL benzonase, 30 min at 37°C, MilliporeSigma catalog # E8263-25KU), and then cell debris was cleared with low-speed spin (1500 g 10 min). The supernatant containing virus was concentrated over a 100 kDa molecular weight cutoff Centricon column (MilliporeSigma catalog # Z648043) to a final volume of ~150 μL. For highly purified large-scale preps this protocol was altered so that ten plates were transfected and harvested together at 3 days after transfection, and then the crude virus was purified by iodixanol gradient centrifugation.
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