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FastDigest SanDI (KflI)

5'…G GG W C C C...3'
3'…C C C W GG G...5'

FastDigest buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by overlapping CpG methylation Not inactivated at 80°C in 20 min Recombinant enzyme LO certified

The FastDigest SanDI (KflI) restriction enyzme recognizes GG^GWCCC sites and cuts best at 37°C in 5-15 minutes using universal Yellow Tango Buffer
  
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Lambda DNA digested with SanDI (KflI)

Linearized pJET1 DNA with inserted SanDI recognition sites, 0.7% agarose, 3 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
Formulation1 µL of enzyme (1 FDU) cleaves 1 μg of linearized pJET1 DNA with inserted SanDI recognition sites 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 SanDI (KflI) is formulated to digest up to:

  • 1 µg of linearized pJET1 DNA with inserted SanDI recognition sites 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 minutes 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
(pJET1-SanDI DNA/BamHI)
5 5 10 5 No
(chloroform extraction)
16

Methylation Effects

  • Dam: never overlaps - no effect.
  • cm: may overlap - effect not determined.
  • CpG: may overlap - blocked.
  • EcoKI: never overlaps - no effect.
  • EcoBI: never overlaps - no effect.

Number of recognition sites in DNA molecules

Methylation type Sequence Cleavage effect
CpG5'...GGGWCCm5C G...3'
3'...CCCWGG Gm5C...5'
Blocked
Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
1 0 0 0 0 0
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
0 0 0 0 0 0

New sites generated by ligation

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

Recognition SequenceSecond EnzymeEnzymes recognizing newly generated recognition sequence
GG^GACCC
  • CpoI (RsrII)/FastDigest RsrII (CpoI)* (CG^GACCG)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (G^GACC)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • FaqI (BsmFI)/FastDigest BsmFI (FaqI)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RG^GACCC)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • EcoO109I (DraII)/FastDigest EcoO109I
  • FaqI (BsmFI)/FastDigest BsmFI (FaqI)
  • FastDigest SanDI (KflI)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RG^GACCT)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • EcoO109I (DraII)/FastDigest EcoO109I
  • FaqI (BsmFI)/FastDigest BsmFI (FaqI)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)
  • SetI
GG^GTCCC
  • CpoI (RsrII)/FastDigest RsrII (CpoI)* (CG^GTCCG)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (G^GTCC)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RG^GTCCC)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • EcoO109I (DraII)/FastDigest EcoO109I
  • FastDigest SanDI (KflI)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RG^GTCCT)
  • BmgT120I
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • Eco47I (AvaII)/FastDigest AvaII (Eco47I)
  • EcoO109I (DraII)/FastDigest EcoO109I
  • Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)

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

Recognition SequenceNewly generated sequence after reactionEnzymes that cleave the newly generated sequence
GG^GACCCGGGACGACCC
  • [FaqI (BsmFI)/FastDigest BsmFI (FaqI)]