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FastDigest PacI

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

FastDigest buffer for 100% activity Optimal incubation at 37°C Thermal inactivation at 65°C in 10 min Recombinant enzyme LO certified

The FastDigest PacI restriction enzyme recognizes TTAAT^TAA sites and cuts best at 37°C in 5-15 minutes using universal Yellow Tango Buffer
  
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Lambda DNA digested with PacI

pJET1 DNA/SdaI fragments with inserted PacI recognition site, 1.0% agarose, 2 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
Recommended Reaction Conditions

1X FastDigest buffer or 1X FastDigest Green buffer at 37°C.
1 µL of FastDigest PacI is formulated to digest up to:

  • 1 µg of linearized pJET1 DNA with inserted PacI recognition site 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 5 minutes or 5 µg of genomic DNA in 30 minutes
Formulation1 µL of enzyme (1 FDU) cleaves 1 μg of linearized pJET1 DNA with inserted PacI recognition site in 5 minutes at 37°C in 1X FastDigest Buffer.
IsoschizomersSearch for commercial isoschizomers using REsearch.

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 no sites
(pJET1-PacI DNA/SdaI,
5 min)		 5 5 5 2 65°C, 10 min 16

Methylation Effects

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

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
0 0 1 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
TTAAT^TAA
  • Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGAT^CG)
  • PvuI/FastDigest PvuI (CGAT^CG)
  • SfaAI (AsiSI)/FastDigest AsiSI (SfaAI) (GCGAT^CGC)
  • FastDigest MseI (SaqAI)
  • Tru1I (MseI)/FastDigest Tru1I