Overview

Inoue Transformation Buffer (TFB) - Inoue Transformation Buffer is used to prepare ultra-competent E. coli cells with transformation efficiencies far above routine CaCl2 or TSS methods.

The product's value is not the ingredient count alone, but the strict order of operations: pH must be set before MnCl2 addition, and the finished buffer must be sterile-filtered rather than autoclaved.

We also have

S30A Buffer · S30B Buffer · GMExpression Buffer catalogue 

Use and Applications

  • Preparation of ultra-competent DH5alpha, TOP10, or related E. coli strains
  • Large-plasmid transformation, ligation recovery, and multi-fragment assembly workflows
  • CRISPR library cloning and low-abundance plasmid rescue
  • Companion workflow for GMExpression TSS users needing higher efficiency

→Buffer Indicator Navigator

Use the GMExpression Buffer Indicator Navigator to find suitable pH ranges that match this buffer system, or start from a specific buffer and search for suitable working pH ranges for the intended experiment.

Specifications

ItemSpecification
Bottle100 mL or 500 mL sterile bottle
Aliquot option10 mL single-use aliquots for small laboratories
Critical labelDO NOT AUTOCLAVE
ShippingFrozen domestic shipment; dry ice for international shipment

Composition

ComponentAmount / concentrationFunction
PIPES10 mM; CAS 5625-37-6Buffering at pH 6.7
KCl250 mM; CAS 7447-40-7High potassium ionic strength
CaCl2.2H2O15 mM; CAS 10035-04-8Competence induction
MnCl2.4H2O55 mM; CAS 13446-34-9Inoue-method transformation enhancer
KOH / HClq.s. to pH 6.7pH adjustment before MnCl2 addition

Instruction

1Thaw on ice immediately before competent-cell preparation.
2Use in the Inoue protocol with cold-grown E. coli culture, chilled centrifugation, and DMSO addition at the final resuspension step.
3Keep buffer cold throughout use.
4Do not autoclave or heat-sterilise.

Note

  • Any brown discoloration indicates manganese oxidation or precipitation and should be treated as a failed unit.

Cautions

Caution. Autoclaving destroys transformation activity by altering manganese chemistry.
Caution. Do not use aliquots that have undergone more than three freeze-thaw cycles.

Storage and Expiry · Safety

  • Store at -20 deg C. Thaw on ice.
  • Unopened shelf life: 12 months at -20 deg C after QC validation.
  • Transport. Ships frozen on dry ice or gel ice; transfer to the stated storage temperature immediately on receipt and minimise freeze-thaw cycles.
  • Handling and PPE. Wear a laboratory coat, nitrile gloves, and safety glasses for routine handling. Contains manganese and PIPES; avoid ingestion and eye contact. Consult the Safety Data Sheet before use.

References

  1. Inoue, Nojima and Okayama (1990), Gene 96:23-28.
  2. Sambrook and Russell, Molecular Cloning, 3rd edition.

Frequently Asked Questions

Q1. How is this different from TSS?
TSS is a convenient one-step routine transformation buffer. Inoue TFB is a higher-efficiency competent-cell preparation system for difficult cloning.
Q2. Why is the pH order important?
Mn2+ can precipitate as manganese hydroxide if the solution is adjusted near neutral or alkaline pH after MnCl2 addition.