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FastDigest Fnu4HI (SatI)

5'...G C↓N G C...3'
3'...C G N↑C G...5'

FastDigest buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by overlapping CpG methylation Thermal inactivation at 65°C in 5 min LO certified

The FastDigest Fnu4HI (SatI) restriction enzyme recognizes GC^NGC sites and cuts best at 37°C in 5-15 minutes using universal Yellow Tango Buffer (Isoschizomers: Fnu4HI, Fsp4HI, ItaI)
  
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Lambda DNA digested with Fnu4HI (SatI)

Lambda DNA digested with Fnu4HI (SatI), 1.4% agarose, 380 cleavage sites

Thermo Scientific FastDigest enzymes are an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers.

The universal buffer allows rapid single-, double- or multiple DNA digestion within 5-15 minutes eliminating any need for buffer change or subsequent DNA clean-up steps. DNA modifying enzymes, such as Klenow Fragment, T4 DNA Ligase, alkaline phosphatases and T4 DNA Polymerase all have 100% activity in FastDigest Buffer. Therefore, enzymes for downstream applications can be directly added to the FastDigest reaction mix.

For additional convenience FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading of digestion reaction products on gels.

Short incubation times and optimal composition of the universal FastDigest Buffer eliminate star activity effects.

Features

  • 100% activity of all FastDigest enzymes in the universal buffer
  • 100% buffer compatibility with downstream applications
  • Complete digestion in 5-15 minutes
  • Direct loading on gels
  • No star activity
  • 176 FastDigest enzymes available

Applications

  • Molecular cloning
  • Restriction site mapping
  • Genotyping
  • Southern Blot
  • Restriction fragment length polymorphism (RFLP)
  • SNP analysis

Note

At least two copies of FastDigest Fnu4HI (SatI) recognition site are required for efficient cleavage.

The FastDigest Green Buffer offers the same high performance in DNA digestion and downstream applications as the colorless FastDigest Buffer. For applications that require product analysis by fluorescence excitation (e.g. concentration measurements in UV light) the colorless FastDigest Buffer is recommended.

For methylation sensitivity refer to product specifications.

  
Storage Condition-20 C
HazardousNo
Compatible Ends
Formulation1 μL of enzyme (1 FDU) cleaves 1 μg of lambda DNA in 5 minutes at 37°C in 1X FastDigest Buffer.
IsoschizomersSearch for commercial isoschizomers using REsearch.
Recommended Reaction Conditions

1X FastDigest buffer or 1X FastDigest Green buffer at 37°C.
1 µL of FastDigest Fnu4HI (SatI) is formulated to digest up to:

  • 1 µg of lambda DNA in 5 minutes
  • 1 µg of plasmid DNA in 5 minutes
  • 0.2 µg of PCR product in 10 minutes
  • 1 µg of genomic DNA in 5 minutes or 5 µg of genomic DNA in 30 minutes

Reaction conditions

Reaction temperature Digestion time with 1 µL of FastDigest enzyme, min bp from end of DNA required for complete digestion Thermal inactivation Incubation time without star activity, hours
Lambda, 1 µg/20 µL Plasmid DNA, 1 µg/20 µL PCR product, ~0.2 µg/30 µL Genomic DNA, 1 µg/10 µL
37°C 5 5 10 5 3 65°C, 5 min 16

Methylation Effects

  • Dam: never overlaps - no effect.
  • Dcm: never overlaps - no effect.
  • CpG: may overlap - blocked.
  • EcoKI: never overlaps - no effect.
  • EcoBI: never overlaps - no effect.
Methylation type Sequence Cleavage effect
CpG5'...Gm5C GGC...3'
3'...C Gm5CCG...5'		 Blocked
CpG5'...GCNGm5C G...3'
3'...CGNC Gm5C...5'		 Blocked

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
380 31 17 42 19 19
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
20 20 23 23 19 37

New sites generated by ligation

Newly generated recognition sites resulting from fill-in of 5'-overhang and self-ligation

Recognition Sequence Newly generated sequence after reaction Enzymes that cleave the newly generated sequence
GC^NGCGCNNGC
  • Cac8I
GC^AGCGCAAGC
  • Cac8I
GC^CGCGCCCGC
  • Cac8I
  • FauI
  • [SsiI (AciI)/FastDigest AciI (SsiI)]
GC^GGCGCGGGC
  • Cac8I
GC^TGCGCTTGC
  • Cac8I