I want to digest my PCR product with XmnI Enzyme. Package contains : XmnI 1000 units (0.05 ml), 10 X Buffer 4, and BSA 10. Detail is in here : http://66.155.211.155/nebecomm/products_intl/productR0194.asp .
The protocol is not understandable enough (for me). If I want to digest 15 ul of PCR product with these reagents, what is the best condition should I prepare ?
Your BSA is a 100x stock solution. Make a 10x stock solution by diluting in nucelase-free water. Purify our PCR product using a standard kit. Set up a digestion mix (usually 20-50 µl total, a small volume ) containing PCR product, 10% v/v buffer, 10% v/v BSA (from 10x stock), 0,5µl-1µl enzyme, depends on DNA concentration. Incubate best in a water bath. Seal the lid with parafilm. Hope this helps.
Thank you very much. But the package is to small. 1 ul contains 200 units. So, when I use 0,1 ul its already 20 Units. Its enough. But it will be a trouble for a lot of sample caused by pipetting error.
clean up your PCR product with any PCR clean up kit, e.g. Qiagen, Macherey&Nagel, Roche. Elute in water or buffer. For setup of a 20µl digestion take: 15 µl purified PCR product, 2µl Puffer 4, 2µl 10x BSA (ditute the original 100x stock solution, which comes with the enzyme), add 1µl Enzyme. Easy. Incubate at least 2 h at 37 degree. Overnight is also possible.
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