Restriction Enzyme Digest Calculator

Single digest uses one restriction enzyme to cut DNA at specific recognition sites. Double digest uses two enzymes simultaneously, requiring compatible buffer conditions and often longer incubation times for complete digestion.

Generally, use 1-5 units of enzyme per μg of DNA. For double digests, ensure both enzymes have sufficient activity in the chosen buffer system. Overdigestion can occur with excessive enzyme amounts.

BSA (Bovine Serum Albumin) stabilizes enzymes, prevents adhesion to tube walls, and can enhance enzyme activity. Some enzymes require BSA for optimal performance, while others benefit from its presence.

Key factors include DNA purity, enzyme quality, buffer compatibility, incubation temperature and time, DNA concentration, and the presence of enzyme inhibitors or contaminants in the sample.

Select a buffer that supports high activity for both enzymes. Universal buffers work for most combinations, but check manufacturer compatibility charts. Some enzymes may require sequential digestion with different buffers.

Yes, overnight incubation (12-16 hours) at appropriate temperature often ensures complete digestion, especially for difficult templates or double digests. However, some enzymes may cause star activity with prolonged incubation.

Check enzyme activity and expiration, verify buffer compatibility, increase incubation time or temperature, add fresh enzyme, or check for inhibitors in your DNA preparation. DNA methylation can also block some enzymes.

Maintain the same ratios of all components while adjusting volumes proportionally. Keep enzyme concentration consistent (units per μg DNA) and ensure minimum reaction volumes recommended by the manufacturer.