mRNA GMP Enzymes

BspQI (10 U/μl, GMP Grade), SKU: GMP4302PC The engineered mutant of T4 RNA ligase 2 is mainly used for the ligation reaction at the nick sites in the double-stranded RNA (dsRNA) structure.

PRODUCT OVERVIEW

This product, BspQI, is a recombinant protein encoded by the BspQI gene of Bacillus sphaericus and expressed in E. coli. It is a type IIS restriction endonuclease with the following recognition sequence:

5'······GCTCTTC(N)··············3'

3'······CGAGAAG(NNNN)······5'

This product is a GMP-grade recombinant BspQI. During production, strict control is exercised over host proteins, exogenous DNA, RNases, and other process-related impurities, as well as microbial content and bacterial endotoxins. The entire production process does not use or add ampicillin or any animal-derived raw materials or excipients. Production and quality management are carried out in accordance with GMP standards, ensuring traceability of the production process, raw materials, and excipients. The product meets the requirements for raw materials and excipients in the mRNA vaccine production field and other fields.

Performance

See the digestion performance of BspQI (10 U/μl, GMP Grade), SKU: GMP4302PC, in Figure 1 above.

Frequently Asked Questions (FAQ)

Q1. What should be noted during digestion?

A1. Key recommendations:

1. Keep enzyme volume ≤10% of the total reaction volume.

2. Heat inactivation: 80°C for 20 min.

3. Assess plasmid linearization by agarose gel electrophoresis, HPLC, or CE.

4. Use phenol-chloroform extraction if RNase removal is required.

Q2. What may cause incomplete plasmid digestion?

A2. Possible causes and solutions:

1. Suboptimal reaction conditions: Optimize enzyme-to-DNA ratio or extend digestion time.

2. DNA contamination: Replace high-salt buffers with RNase-free water; remove impurities using phenol-chloroform extraction.

3. Enzyme inactivation: Store at -20°C, aliquot before use, and avoid repeated freeze-thaw cycles.

4. Improper enzyme dilution: Use storage buffer for dilution.

5. Missing recognition site: Confirm plasmid sequence by Sanger sequencing.

6. High open-circle content: Optimize plasmid prep to increase supercoiled DNA (avoid vigorous mixing).

7. Plasmid multimers: Use an appropriate host strain or increase enzyme amount.

Q3. Why do smeared bands appear after digestion?

A3. Possible causes and solutions:

1. Poor DNA quality: Use suitable host strains or perform phenol-chloroform extraction.

2. Reagent contamination: Use fresh running buffer and clean agarose gels.

3. Excess enzyme: Reduce enzyme amount and re-optimize conditions.