Description

Biofargo XhoI, GMP Grade is a restriction endonuclease produced by recombinant expression in E. coli and manufactured under GMP specifications, for use as a raw material in mRNA, plasmid and vaccine production. XhoI cuts the palindromic site CTCGAG, leaving a four-base 5′ overhang. It is a common backbone site for plasmid linearisation and is unaffected by Dam and Dcm methylation, though CpG methylation impairs cleavage.

Manufacturing is fully traceable through the process and raw materials. No antibiotics, animal-derived materials or animal-derived excipients are used anywhere in the process. Process-related impurities are controlled and released against specification: host cell protein, residual host DNA, non-specific endonuclease, DNase and RNase activity, microbial limits, bacterial endotoxin, mycoplasma and heavy metals.

The enzyme digests target DNA in 15 minutes to 1 hour at 37 °C and is heat-inactivated at 80 °C for 20 minutes. It is supplied at 20 U/µL with six vials of the matching 10× Cut Buffer F, GMP Grade, so no buffer has to be ordered separately. A Certificate of Analysis is issued for every lot.

For Research Use Only unless qualified by the customer for its intended manufacturing use. Not for use in diagnostic or therapeutic procedures.

Specifications

Parameter	Specification
Product Name	Biofargo XhoI, GMP Grade
Catalog Number	BF-E4004
Grade	GMP Grade
Enzyme Type	Type II restriction endonuclease
Recognition / Cut Site	5′…C T C G A G…3′ 3′…G A G C T C…5′
Isoschizomers	PaeR7I, Sfr274I, SlaI (methylation sensitivities may differ)
Concentration	20 U/µL
Pack Size	20,000 U (1 mL)
Supplied Buffer	10× Cut Buffer F, GMP Grade, 6 × 1 mL
Reaction Temperature	37 °C
Digestion Time	15 minutes to 1 hour
Heat Inactivation	80 °C for 20 minutes
Unit Definition	Completely digest 1 µg of λDNA (HindIII digest) in 1 hour at 37 °C in a 50 µL reaction
Protein Purity	≥95% by SDS-PAGE (Coomassie Blue)
Host Cell Protein	<50 ppm (ChP 2025 Vol. IV, General Chapter 3412)
Residual Host DNA	<10 copies/20 U (ChP 2025 Vol. IV, General Chapter 3407, qPCR)
Bacterial Endotoxin	<0.5 EU/KU (ChP 2025 Vol. IV, General Chapter 1143, gel-clot)
Microbial Limit	Total aerobic count <5 cfu/mL; yeasts and moulds <5 cfu/mL (ChP 2025 Vol. IV, General Chapter 1105)
Mycoplasma	Negative (LAMP)
Heavy Metals	<10 ppm (ChP 2025 Vol. IV, General Chapter 0821)
Animal-Derived Components	None
Antibiotics	None used in manufacture
Storage	−20 ± 5 °C, valid 24 months
Shipping	≤0 °C (blue ice)
Documentation	Lot-specific Certificate of Analysis

Features

GMP manufacture with full traceability of process and raw materials, for use as a raw material in mRNA, plasmid and vaccine production.
No animal-derived components and no antibiotics anywhere in the process, which removes two of the questions a raw-material compliance review always asks.
Impurities released against specification — host cell protein, residual host DNA, non-specific endonuclease, DNase, RNase, microbial limits, endotoxin, mycoplasma and heavy metals, each by a stated pharmacopoeial method.
Buffer included — six 1 mL vials of 10× Cut Buffer F, GMP Grade ship with the enzyme, so the digest does not depend on a separate buffer order.
Fast, complete digestion in 15 minutes to 1 hour, with heat inactivation at 80 °C for 20 minutes.
Lot-specific Certificate of Analysis with every shipment.
24-month shelf life at −20 ± 5 °C.

Applications

Plasmid linearisation for IVT: Linearise the DNA template before in vitro transcription, where template integrity and a defined 3′ end set the transcript length and translation efficiency.
mRNA manufacture: Supply the upstream digestion step as a GMP-grade raw material with documented impurity control.
Plasmid DNA production: Linearise and verify plasmid constructs under a controlled process.
Vaccine and cell therapy manufacture: Use as a raw material where animal-component-free and antibiotic-free sourcing has to be demonstrated.
Process development: Run the same enzyme at research scale and at manufacturing scale without changing supplier or revalidating.

Kit Components

Component	Volume	Storage
XhoI, GMP Grade (20 U/µL)	20,000 U (1 mL)	−20 ± 5 °C
10× Cut Buffer F, GMP Grade	6 × 1 mL	−20 ± 5 °C

Storage & Shipping

Ship at ≤0 °C on blue ice.
Store at −20 ± 5 °C. Valid for 24 months.
Avoid repeated freeze-thaw cycles; keep the enzyme on ice while setting up reactions.
A lot-specific Certificate of Analysis is supplied with every shipment.

Protocol Overview

Combine on ice, in order: nuclease-free water to 50 µL, 5 µL of 10× Cut Buffer F, GMP Grade, 1 µg of DNA, and 1 µL of XhoI, GMP Grade (20 U/µL).
Mix gently and spin down.
Incubate at 37 °C for 15 minutes to 1 hour.
Inactivate by heating at 80 °C for 20 minutes.
Keep the enzyme below 10% of the total reaction volume to avoid star activity from carried-over glycerol.

Frequently Asked Questions (FAQs)

Common questions about Biofargo XhoI, GMP Grade.

What does GMP Grade mean for this enzyme?

It is manufactured and released under GMP specifications, with full traceability of the process and of the raw materials, and with process-related impurities controlled to a written specification. Host cell protein, residual host DNA, non-specific endonuclease, DNase and RNase activity, microbial limits, bacterial endotoxin, mycoplasma and heavy metals are each tested by a stated pharmacopoeial method and reported on the lot Certificate of Analysis.

Are animal-derived materials or antibiotics used?

No. Neither animal-derived materials or excipients nor antibiotics are used anywhere in the manufacturing process. This is normally the first question in a raw-material compliance review, so it is stated on the Certificate of Analysis.

What impurity levels are specified?

Protein purity is ≥95% by SDS-PAGE. Host cell protein is <50 ppm, residual host DNA <10 copies/20 U, bacterial endotoxin <0.5 EU/KU, heavy metals <10 ppm, total aerobic microbial count <5 cfu/mL with yeasts and moulds <5 cfu/mL, and mycoplasma negative by LAMP.

How is a digest set up?

On ice, combine 5 µL of 10× Cut Buffer F, GMP Grade, 1 µg of DNA, 1 µL of enzyme and nuclease-free water to 50 µL. Mix gently, spin down, incubate at 37 °C for 15 minutes to 1 hour, then inactivate at 80 °C for 20 minutes.

Why should the enzyme stay below 10% of the reaction volume?

The enzyme storage buffer contains glycerol. Above about 10% of the total reaction volume, the carried-over glycerol can cause star activity. The same logic applies in reverse to contaminants in the DNA: the smaller the reaction, the more strongly salt, EDTA or ethanol in the substrate inhibits digestion.

What can interfere with the digest?

DNA substrates should carry no phenol, chloroform, ethanol, EDTA, detergent or high salt. All of these reduce enzyme activity, and their effect is proportionally worse in a small reaction volume.

Is cleavage affected by methylation?

Partly. Dam methylation: no effect; Dcm methylation: no effect; CpG methylation: impaired; EcoKI methylation: no effect; EcoBI methylation: no effect. The table on this page gives the full picture; check it against the host the plasmid was grown in.

Does it come with buffer?

Yes. Six 1 mL vials of 10× Cut Buffer F, GMP Grade ship with every unit, so the digest does not depend on ordering a buffer separately.

How is it stored and shipped?

It ships at ≤0 °C on blue ice and is stored at −20 ± 5 °C, where it is valid for 24 months.

Can you support a DMF filing or a custom GMP enzyme?

Yes. Drug Master File support and custom GMP enzyme manufacture can be arranged. Contact us at contact@biofargo.com with the enzyme, the scale and the regulatory route you need.

Do you provide a Certificate of Analysis?

Yes. A lot-specific Certificate of Analysis is issued with every shipment and can be sent in advance for a raw-material qualification review. Request it at contact@biofargo.com.

Can it be used for clinical or diagnostic purposes?

No. This product is for research use only and is not intended for human, diagnostic or therapeutic use.

Methylation Effects on Digestion

Dam	Dcm	CpG	EcoKI	EcoBI
No effect	No effect	Impaired	No effect	No effect

Number of Recognition Sites in Common DNA

λDNA	ΦX174	pBR322	pUC57	pUC18/19	SV40	M13mp18/19	Adeno2
1	1	0	0	0	0	0	6

Related Products

The Biofargo GMP-grade restriction endonuclease range covers both scarless Type IIS sites and the conventional backbone sites, so a change of plasmid backbone does not mean a change of supplier or a second raw-material qualification.

Biofargo BsaI, GMP Grade — Type IIS, scarless cleavage; 20 U/µL, 20,000 U.
Biofargo XbaI, GMP Grade — Type II, palindromic site; 20 U/µL, 20,000 U.
Biofargo BspQI, GMP Grade — Type IIS, scarless cleavage; 10 U/µL, 10,000 U.

Documents

Request Datasheet