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FastDigest EcoRI

5'...GA A T T C...3'
3'...C T T A AG...5'
Available On-Site

FastDigest buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by overlapping CpG methylation Thermal inactivation at 80°C in 5 min Recombinant enzyme LO certified

FastDigest EcoRI
The FastDigest EcoRI restriction enzyme recognizes G^AATTC sites and cuts best at 37°C in 5-15 minutes using universal FastDigest Buffer
  
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Lambda DNA digested with EcoRI

Lambda DNA digested with EcoRI, 0.7% agarose, 5 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)

Note

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.

  
Compatible EndsFastDigest MfeI (MunI), FastDigest Tsp509I (TasI), FastDigest XapI, MfeI, MunI, TasI, XapI.
Formulation1 μL of enzyme (1 FDU) cleaves 1 μg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
HazardousNo
IsoschizomersSearch for commercial isoschizomers using REsearch.
Reaction Conditions

1X FastDigest buffer or 1X FastDigest Green buffer at 37°C.

  • 1 µL of FastDigest EcoRI is formulated to digest up to:
  • 1 µg of lambda DNA in 5 minutes
  • 1 µg of plasmid DNA in 15 minutes
  • 0.2 µg of PCR product in 20 minutes
  • 1 µgof genomic DNA in 5 minutes or 5 µ of genomic DNA in 30 minutes
Storage Condition-20 C

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 15 20 5 2 80°C, 5 min 0.5

Methylation Effects

Methylation type Sequence Cleavage effect
CpG5'...m5C GAATTm5C G...3'
3'... Gm5CTTAA Gm5C...5'
Impaired
EcoBI(TGA(N)8TGCT)5'...TGm6AATTC(N)4 TGCT...3'
3'...AC TTAAG(N)4m6ACGA...5'
No effect

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
5 0 1 1 1 1
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
1 1 1 1 0 1

New sites generated by ligation

Newly generated recognition sites resulting from ligation of protruding compatible DNA ends

Recognition Sequence Second Enzyme Enzymes recognizing newly generated recognition sequence
G^AATTC
  • MunI (MfeI)/FastDigest MfeI (MunI) (C^AATTG)
  • TasI (Tsp509I)/FastDigest Tsp509I (TasI) (^AATT)
  • TasI (Tsp509I)/FastDigest Tsp509I (TasI)
  • XapI (ApoI)/FastDigest XapI* (R^AATTC)
  • EcoRI/FastDigest EcoRI
  • TasI (Tsp509I)/FastDigest Tsp509I (TasI)
  • XapI (ApoI)/FastDigest XapI
  • XapI (ApoI)/FastDigest XapI* (R^AATTT)
  • TasI (Tsp509I)/FastDigest Tsp509I (TasI)
  • XapI (ApoI)/FastDigest XapI

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
G^AATTCGAATTAATTC
  • PdmI (XmnI)/FastDigest PdmI
  • FastDigest MseI (SaqAI)
  • [TasI (Tsp509I)/FastDigest Tsp509I (TasI)]
  • Tru1I (MseI)/FastDigest Tru1I
  • VspI (AseI)/FastDigest AseI (VspI)

 

References

Citations