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FastDigest MboI

5'...↓G A T C ...3'
3'... C T A G↑...5'

FastDigest buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by Dam methylation Thermal inactivation at 65°C in 15 min LO certified

The FastDigest MboI restriction enzyme recognizes ^GATC sites and cuts best at 37°C in 5-15 minutes using universal Yellow Tango Buffer (Isoschizomers: BfuCI, BssMI, BstKTI, BstMBI, DpnII, Kzo9I, NdeII, Sau3AI)
  
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Lambda DNA digested with MboI

Lambda DNA (dam-), 1.4% agarose, 116 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

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 FastDigest BamHI, FastDigest BclI, FastDigest BglII, FastDigest PsuI, BclI, BamHI, BglII, PsuI.
Formulation1 μL of enzyme (1 FDU) cleaves 1 μg of lambda DNA dam- 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 MboI is formulated to digest up to:

  • 1 µg of lambda DNA dam- in 5 minutes
  • 1 µg of plasmid DNA in 5 minutes
  • 0.2 µg of PCR product in 5 minutes
  • 1 µg of genomic DNA in 10 min or 5 µg of genomic DNA in 60 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 5 5 2 65°C, 5 min 16

Methylation Effects

Methylation type Sequence Cleavage effect
Dam(GATC)5'...GAAGm6A TC...3'
3'...CTTC Tm6AG...5'		 Blocked
CpG5'...m5C GAAGA...3'
3'... Gm5CTTCT...5'		 No effect
EcoBI(TGA(N)8TGCT)5'...TGm6AAGA(N)5 TGCT...3'
3'...AC TTCT(N)5m6ACGA...5'		 No effect

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
130 11 11 11 7 8
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
9 9 8 8 15 11

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
^GATC
  • BamHI/FastDigest BamHI (G^GATCC)
  • BclI/FastDigest BclI (T^GATCA)
  • BglII/FastDigest BglII (A^GATCT)
  • Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (^GATC)
  • PsuI (BstYI)/FastDigest PsuI* (R^GATCC)
  • PsuI (BstYI)/FastDigest PsuI* (R^GATCT)
  • Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)
  • DpnI/FastDigest DpnI
  • MboI/FastDigest MboI

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
^GATCGATCGATC
  • [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)]
  • Bsu15I (ClaI)/FastDigest ClaI (Bsu15I)
  • [DpnI/FastDigest DpnI]
  • [MboI/FastDigest MboI]
  • TaqI/FastDigest TaqI