EcoRV

Cat. No.	Component	Quantity	Storage	Concentration
MCE1009-S (2.000 unit)	EcoRV	1 x 0.1 ml	-20˚C	20,000 U/ml
	UCut Buffer	1 x 1.5 ml	-20˚C	6X
	Blue Gel Loading Dye	1 x 1.5 ml	25˚C	10X
MCE1009-L (8.000 unit)	EcoRV	4 x 0.1 ml	-20˚C	20,000 U/ml
	UCut Buffer	2 x 1.5 ml	-20˚C	6X
	Blue Gel Loading Dye	2 x 1.5 ml	25˚C	10X

Reagent	Amount
Restriction Enzyme	1 µl*
UCut Buffer	5 µl
DNA	1 µg**
Water (Ultra Pure)	X***
Total Reaction Volume	50 µl

 

Incubate the enzyme reaction at 37 °C for 1 hour.

*When using multiple enzymes for digestion, the total enzyme volume should not exceed 5 µl in a 50 µl reaction.

**The DNA template should be of high purity, devoid of any residual impurities such as phenol or ethanol, which are known to inhibit restriction digestion reaction.

***Ultra-pure water is added to bring the total reaction volume to 50 µl.

Problem 1: No digestion or incomplete digestion

Possible Causes:

• Incorrect enzyme concentration.
• Enzyme inactivation due to improper storage or handling.
• Inadequate incubation time or temperature.
• Inhibitory substances present in the DNA or buffer.

Solutions:

• Check enzyme amount: Ensure that the enzyme amount used is within the recommended range. For multiple enzymes, ensure the total enzyme volume does not exceed 5 µl in a 50 µl reaction.
• Verify enzyme activity: Ensure that the enzyme has been properly stored (usually at -20°C) and has not expired.
• Optimize incubation conditions: Incubate the reaction at the optimal temperature for the specific enzyme (usually 37°C). Some enzymes may require a longer incubation time than recommended (1–2 hours).
• Check the DNA purity: Ensure the DNA template is free of contaminants (such as proteins, RNA, or ethanol), which may inhibit the enzyme activity.

Problem 2: Partial digestion (only some fragments are digested)

Possible Causes:

• Incomplete mixing of reagents.
• Inappropriate reaction conditions.
• High DNA concentration leading to incomplete digestion.

Solutions:

• Ensure proper mixing: Gently mix the reaction components by pipetting or flicking the tube before incubation.
• Check DNA concentration: Avoid using excessive amounts of DNA (typically 1 µg per 50 µl reaction). High DNA concentration may inhibit enzyme action.
• Verify reaction conditions: Make sure the correct buffer. Use fresh enzyme if necessary.

Problem 3: Star activity (non-specific digestion)

Possible Causes:

• Incorrect enzyme storage or handling.
• Excessive enzyme concentration.
• Incorrect reaction conditions (e.g., too high or low temperature, too high salt concentration).

Solutions:

• Check enzyme storage: Ensure enzymes are stored at -20°C and have not been subjected to multiple freeze-thaw cycles.
• Adjust enzyme concentration: Reduce enzyme concentration if it exceeds recommended levels (typically no more than 5 µl in a 50 µl reaction).
• Optimize reaction conditions: Ensure the reaction is carried out in the correct buffer with the appropriate salt concentration. Avoid high salt or alcohol levels, which can cause star activity.

Problem 4: Uneven digestion (uneven bands in agarose gel)

Possible Causes:

• Uneven distribution of enzymes in the reaction mixture.
• DNA degradation or contamination.

Solutions:

• Mix thoroughly: Ensure the enzyme and buffer are well mixed with the DNA.
• Ensure DNA integrity: Check the quality of the DNA template.
• Degraded DNA may result in uneven digestion patterns. Use pure, high-quality DNA for digestion.

Problem 5: Excessive background (bands or smear not related to expected digestion pattern)

Possible Causes:

• Excessive enzyme or buffer contamination.
• Impure DNA template.

Solutions:

• Use fresh reagents: Check for contamination of enzymes, buffers, or water. Use freshly prepared or aliquoted reagents.
• Clean DNA template: Re-purify the DNA to remove any potential contaminants such as salts, proteins, or ethanol.

General Tips:

• Always use freshly thawed enzymes and buffers.
• Keep reactions on ice until ready to begin incubation.
• Check buffer compatibility with the enzymes and make sure the pH and salt concentrations are optimal.
• If troubleshooting persists, try performing the digestion with a smaller amount of DNA or a different batch of enzymes.