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KspAI (HpaI)

5'...G T T↓A A C...3'
3'...C A A↑T T G...5'
Available as a FastDigest enzyme for rapid DNA digestion

FastDigest enzyme available B buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by overlapping CpG methylation Thermal inactivation at 65°C in 20 min Genome qualified LO certified

The KspAI (HpaI) restriction enzyme recognizes GTT^AAC sites and cuts best at 37°C in B buffer (Isoschizomers: HpaI)
  
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Lambda DNA digested with KspAI (HpaI)

Lambda DNA digested with KspAI (HpaI), 0.7% agarose, 14 cleavage sites

Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.

Highlights

  • Superior quality – stringent quality control and industry leading manufacturing process
  • Convenient color-coded Five Buffer System
  • Includes universal Tango buffer for double-digestions
  • BSA premixed in reaction buffers
  • Wide selection of restriction endonuclease specificities

Applications

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

Note

For methylation sensitivity refer to product specifications.

  
Storage Condition-20 C
HazardousNo
Conditions for 100% Activity
  • 1X Buffer B: 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, and 0.1 mg/mL BSA
  • Incubate at 37°C
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours.
Double DigestionPerform double digestion using DoubleDigest.
IsoschizomersSearch for commercial isoschizomers using REsearch.
NoteHigh glycerol (> 5%) concentration, pH > 8.0 or a large excess of enzyme may result in star activity.
Storage BufferKspAI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/mL BSA, and 50% (v/v) glycerol.

Reaction conditions

Recommended buffer for 100% activity Optimal temperature Enzyme activity in Thermo Scientific buffers, % Tango buffer for double digestion
B (blue)

1X		 G (green)

1X		 O (orange)

1X		 R (red)

1X		 Tango

(yellow)

1X / 2X
B (Blue) 37°C 100 50-100† 20-50 20-50 100† 50-100 1X† or 2X†

† Star activity appears at a greater than 5-fold overdigestion (5 units x 1 hour).

Methylation Effects

Methylation type Sequence Cleavage effect
CpG 5'...GTTAAm5C G...3'

3'...CAATT Gm5C...5'		 Impaired
CpG 5'...m5C GTTAAm5C G...3'

3'... Gm5CAATT Gm5C...5'		 Blocked
EcoKI (AAC(N)6GTGC) 5'...GTTAm6AC(N)6G TGC...3'

3'...CAAT TG(N)6Cm6ACG...5'		 Blocked

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1 2 3 4 5
20-50 50-100

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
14 3 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 blunt DNA ends

Recognition sequence Second restriction enzyme Restriction enzymes cleaving the newly generated recognition sequence
GTT^AAC
  • AanI (PsiI)/FastDigest PsiI (AanI) (TTA^TAA)
  • DraI/FastDigest DraI (TTT^AAA)
  • MssI (PmeI)/FastDigest MssI (GTTT^AAAC)
  • SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTT^AAAT)
  • FastDigest MseI (SaqAI)
  • Tru1I (MseI)/FastDigest Tru1I
  • Bsp68I (NruI)/FastDigest NruI (RruI) (TCG^CGA)
  • TaqI/FastDigest TaqI
  • Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTA^TAC)
  • Hpy8I (MjaIV)/FastDigest Hpy8I
  • FspAI/FastDigest FspAI* (RTGC^GCAC)
  • FspAI/FastDigest FspAI* (RTGC^GCAT)
  • NsbI (FspI)/FastDigest FspI (NsbI) (TGC^GCA)
  • HpyCH4V
  • HincII (HindII)/FastDigest HincII* (GTY^AAC)
  • HincII (HindII)/FastDigest HincII
  • Hpy8I (MjaIV)/FastDigest Hpy8I
  • KspAI (HpaI)/FastDigest HpaI (KspAI)
  • FastDigest MseI (SaqAI)
  • Tru1I (MseI)/FastDigest Tru1I
  • HincII (HindII)/FastDigest HincII* (GTY^GAC)
  • HincII (HindII)/FastDigest HincII
  • Hpy8I (MjaIV)/FastDigest Hpy8I
  • RsaI/FastDigest RsaI (GT^AC)
  • ScaI/FastDigest ScaI (AGT^ACT)
  • MaeIII