Restriction enzyme that i used for double digestion is EcoR1 and Xho1 for 1,2 and 3 hours but didnt get digested band on gel.
RE is of Genei company.
Although gel electrophoresis image is not given, from 3 bands we can assume it is okay for plasmid. The following suggestions can be helpful:
1. Please check the enzyme units required for per ug DNA and add accordingly.
2. To ensure the activity of both enzymes, a reaction can be designed with 3 tubes- first one with EcoR1 only, second one with Xho1 only and third one with both enzymes. Tube 1 and 2 can be of smaller reaction volume. If all three tubes show single band, then we can confirm, both enzymes are working.
3. It is recommended to aliquot buffer in small volumes to avoid repeated freezing and thawing.
4. You can try a 100uL reaction volume with an overnight reaction time.
Hope it will work.
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