Semi-prestained Protein Marker
(usage 5μl/lane)

Preparation: Gently mix the semiprestained Protein Marker by vortexing or pipetting up and down several times. Pipette the desired amount (5-7μl/lane) to a separate tube. Incubate at 95-100^oC for 5 minutes, spin down and load.

Storage buffer:  62,5mM Tris-HCl (pH=6,8), 2% SDS, 1mM EDTA, 30% Glycerol, 0,01% Bromophenol blue, 1mM NaN_3, 50mM DTT. Store at -20^oC.

Note:

• Do not boil the aliquots a second time.
• The recommended load is 5-7μl in a gel lane. Loading less amount may result in less intense bands.
• Run gel using constant voltage, according to manufacturer’s specifications
• Run until dye reaches the bottom of the gel, but does not run off

.

 Source: Purified from an E. coli strain carrying a plasmid with the cDNA o RNA polymerase of T7 phage.

Description: MINOTECH T7 RNA polymerase is an efficient RNA polymerase able to transcribe DNA sequences under the T7 phage promoter up to (5.5 kbs).

Unit definition: One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 μl in 1 hour at 37°C.

Quality Control Assays:

• Functional Assay: T7 RNA polymerase has been tested on PCR products, annealed oligos and digested or supercoiled plasmid DNA containing T7 promoter Sequence: TAATACGACTCACTATA. 100 units of enzyme incubated with the various substrates obtaining transcripts ranging from 120-5500 nucleotides. The full length expected transcripts were found to be above 95% pure based on an ethidium bromide-stained agarose gel.
• Absence of contaminants: Tested extensively for the absence of endo-, exodeoxyribonucleases and RNases.

Guaranteed stability: T7 RNA polymerase is guaranteed to maintain stability until expiration date.

 *PCR products/annealed oligos or plasmid DNA containing T7 promoter

• Incubate at 37^οC for 2 hours.
• Optionally add 0,5 -1 u DNAse I and incubate for 15 min at 37^οC to remove input DNA.
• Purify RNAs by standard phenol chloroform protocol described in Maniatis et al., 1982.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, p. 194-197. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y

 Source: Purified from an E. coli strain carrying a plasmid with M-MuLV reverse transcriptase gene.

Description: High purity reverse transcriptase suitable for first strand  cDNA synthesis. 

Unit definition: One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT)18 as template.

Quality Control Assays:

• Functional Assay: MINOTECH RT is tested for performance in first strand cDNA synthesis followed by PCR with Taq polymerase. The resulting 1.600 bp PCR product is visualized as a single band on an ethidium bromide-stained agarose gel. MINOTECH RT has been successfully used for synthesis of DNA fragments up to 8.8kb size.
• Absence of contaminants: Tested extensively for the absence of nicking, endo- and exodeoxyribonucleases and RNases

Guaranteed stability: MINOTECH RT is guaranteed to maintain stability until expiration date.

Recommended First-Strand cDNA Synthesis mixture:

1. Heat mixture to 65°C for 5 minutes
2. Incubate on ice for at least 1 minute
3. Brief centrifugation and add:

* 400 U of MINOTECH RT can be added to increase yield (for the generation of cDNA >5kb).

4. Mix gently. If using random primers, incubate tube at 25°C for 5 minutes.
5. Incubate at 37-42°C for 30–60 minutes.
6. Heat inactivation step at 70°C for 15 minutes.

Optional (recommended for PCR targets >1kb). Remove RNA complementary to the cDNA, by adding 2 units of E. coli RNase H and incubate at 37°C for 20 minutes.

XbaI is a restriction enzyme purified from Xanthomonas badrii.

Unit substrate: Lambda DNA (dam^-/HindIII digest).

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 200 units of XbaI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNAdam^-/HindIII digest at 37^oC. After 100-fold overdigestion with XbaI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 100 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol , 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Blocked by overlapping

dcm methylation: Not sensitive

Reagents supplied: 10x M and 10x K buffer

TaqI is a restriction enzyme purified from Thermus aquaticus YT I.

Unit substrate: Lambda DNA (dam^-).

Unit calculation assay conditions: 100 mM KCl, 20 mM Tris-HCl (pH 8.5 @ 25°C), 3 mM MgCl₂, 0.04% Triton X-100, 100 μg/ml BSA. Incubate at 65^oC.

Absence of contaminants: 100 units of TaqI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA (dam^-) at 65^oC. After 50-fold overdigestion with TaqI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 300 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA and 50% glycerol. Store at -20^oC

Heat inactivation: 80^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Blocked by overlapping

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x U_TaqI buffer

StyI is a restriction enzyme purified from E.coli WA921/pST27 hsd+.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 50 units of StyI do no produce any unspecific clevage products after 16 hrs incubation with 1 μg of λ DNA at 37°C. After 50-fold overdigestion with StyI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x H buffer

SstI

(SacI isoschizomer)

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl2, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 100 units of SstI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/HindIII digest at 37^oC. After 50-fold overdigestion with SstI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x L and 10x K buffer

CspAI is a restriction enzyme purified from Corynebacterium species.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 10 mM Bis Tris Propane-HCl (pH 7.0 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 50 units of CspA I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 10-fold overdigestion with CspA I, greater than 90% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 100 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x U_CspAI and 10x K buffer

BstEII is a restriction enzyme purified from Bacillus stearothermophilus.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM KCl, 10 mM Tris-HCl (pH 7.4 @ 25°C), 5 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100, 100 μg/ml BSA. Incubate at 60^oC.

Absence of contaminants: 150 units of BstEII do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 60^oC. After 100-fold overdigestion with BstEII, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 100 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Note: BstE II exhibits 10-15% activity at 37°C.

Reagents supplied: 10x U_BstEII and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 30 units of SspI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37°C. After 10-fold overdigestion with SspI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50%  glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x H and 10x K buffer

BssAI is a restriction enzyme purified from Bacillus species.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM KCl, 20 mM Tris-HCl (pH 8.5 @ 25^oC), 3 mM MgCl₂, 0.04% Triton X-100, 100 μg/ml bovine serum albumin and DNA. Incubate at 65^oC.

Absence of contaminants: 50 units of BssA I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 65^oC. After 30-fold overdigestion with BssA I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reference: Rina, M., Stratidakis, I. and Bouriotis, V. (1990). Nucleic Acids Res. 18, 6161.

Reagents supplied: 10x U_BssAI and 10x K buffer

SseBI

(Stu I isoschizomer)

SseBI is a restriction enzyme purified from Streptomyces species.

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl2, 1 mM dithiothreitol, 100 μg/ml BSA and DNA. Incubate at 37^oC.

Absence of contaminants: 150 units of SseBI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/Hind III digest at 37^oC. After 50-fold overdigestion with SseBI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Blocked by overlapping

CpG methylation: Not sensitive

Reagents supplied: 10x H and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 50 units of SphI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37^oC. After 10-fold overdigestion with SphI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 100 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 400 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x M and 10x K buffer

BshF I

(Hae III isoschizomer)

BshFI is a restriction enzyme purified from Bacillus sphaericus.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 200 units of BshF I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 50-fold overdigestion with BshF I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 80^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reference: Vlatakis, G., Clark, D. and Bouriotis, V. (1989). Nucleic Acis Res. 17, 8882

Reagents supplied: 10x A and 10x K buffer

BseCI is a restriction enzyme purified from Bacillus stearothermophilus.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl  (pH 7.9 @ 25°C), 10  mM  MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 55^oC.

Absence of contaminants: 150 units of BseCI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 55^oC. After 100-fold overdigestion with BseCI greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 100 mM KCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Blocked by overlapping

dcm methylation: Not sensitive

CpG methylation: Blocked

Reference: Rina, M., Dialektakis, D., Clark, D., Pagomenou, M. and Bouriotis, V. (1992). Nucleic Acids Res. 20

Reagents supplied: 10x H and 10x K buffer

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25°C), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 25^oC.

Absence of contaminants: 150 units of SmaI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA (HindIII digest) at 25^oC. After 50-fold overdigestion with SmaI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Reagents supplied: 10x A and 10x K buffer

Sla I

(Xho I isoschizomer)

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 150 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 400 units of Sla I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA (Hind III digest) at 37^oC. After 100-fold overdigestion with SlaI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

Reagents supplied: 10x SH and 10x K buffer

SgrBI

(SacII isoschizomer)

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 10 mM Tris-HCl (pH 7.9@ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 400 units of SgrB I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA (HindIII digest) at 37°C. After 100-fold overdigestion with SgrBI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50%  glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Note: Particular sites in λ and φΧ174 DNAs are difficult to cleave with SgrB I, as well as with its prototype Sac II.

Reference: Rina, M., Pagomenou, M. and V, Bouriotis (1991) Nucleic Acids Res. 19, 6342.

Reagents supplied: 10x U_SgrBI and 10x K buffer

Unit substrate: Adenovirus-2 DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 50°C.

Absence of contaminants: 100 units of Sfi I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Adeno-2 DNA at 50^oC. After 50-fold overdigestion with Sfi I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 300 mM NaCl, 5 mM KPO₄  (pH 7.4), 0.1 mM  EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Impaired by overlapping

CpG methylation: Blocked by some combinations of overlapping

Reagents supplied: 10x M and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM KCl, 10 mM Bis-Tris-Propane (pH 7.0 @ 25°C), 10 mM MgCl₂, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 600 units of Sca I incubated for 16 hours at 37ºC with 1 μg of λ DNA resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. After 100-fold overdigestion with Sca I, greater than 90% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 80^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Star activity: Large excess of the enzyme may results in the appearance of star activity.

Reagents supplied: 10x U_ScaI buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.4 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 200 units of Pst I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 100-fold overdigestion with Pst I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 200 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin, 0.15% Triton X-100 and 50% glycerol. Store at -20^oC.

Heat inactivation: 80^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Star activity: Conditions of high enzyme concentration or glycerol concentration>12% may result in star activity.

Reagents supplied: 10x U_PstI and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 50 units of Sau3A I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37^oC. After 50-fold overdigestion with Sau3A I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by overlapping

Reagents supplied: 10x M and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 25^oC.

Absence of contaminants: 80 units of PspP I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 25^oC. After 50-fold overdigestion with PspP I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 55^oC for 15 minutes..

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Blocked by overlapping

CpG methylation: Blocked by overlapping

Note: Incubation at 37^oC results in 60% activity.

Reference: Rina, M., Caufrier, F., Mavromatis, K., Markaki, M., Kokkinidis, M. and Bouriotis, V. (1997) Gene, 197, 353-360.

Reagents supplied: 10x M and 10x K buffer

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 150 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 400 units of Sal I incubated for 16 hours at 37ºC with 1 μg of λ DNA (HindIII digest) resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. After 50-fold overdigestion with Sal I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 300 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Star activity: Large excess of the enzyme results in the appearance of star activity.

Reagents supplied: 10x SH buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM KCl, 50 mM Tris-HCl (pH 8.0 @ 25^oC), 10 mM MgCl₂, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 80 units of Nru I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 10-fold overdigestion with Nru I, less than 20% of the DNA fragments can be ligated.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Blocked by overlapping

dcm methylation: Not sensitive

CpG methylation: Blocked

Star activity: Large excess of the enzyme results in the appearance of star activity.

Reagents supplied: 10x U_NruI and 10x K buffer

NotI is a restriction enzyme purified from Nocardia otitidis-caviarum.

Unit substrate: Adenovirus-2 DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 5 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 80 units of Not I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Adeno-2 DNA at 37°C. After 30-fold overdigestion with Not I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 500 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Note: Supercoiled plasmids may require up to 5-fold more Not I for complete digestion than linear DNAs.

Reagents supplied: 10x U_NotI  buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 400 units of Rsa I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37^oC.  After 10-fold overdigestion with Rsa I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

Note: Cleaves single-stranded DNA slowly.

Reagents supplied: 10x M and 10x K buffer

Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25°C), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 80 units of NheI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/Hind III digest at 37^oC. After 100-fold overdigestion with NheI, greater than 98% of the DNA fragments can be ligated and recut.

Storage buffer: 200 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65°C for 20 minutes..

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

Note: Activity inhibited by salt concentrations >100mM. Cleaves to leave a 5’ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI, or XbaI.

Star activity: Low salt, high glycerol (>5%) concentrations, pH >8.0 or large excess of the enzyme may result in star activity.   

Reagents supplied: 10x A and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl₂, 1 mM DTT, 0.02% Triton X-100, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 100 units of Nco I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 50-fold overdigestion with Nco I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x U_NcoI and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 100 units of Pvu II do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 10-fold overdigestion with Pvu II greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin  and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5% or pH >8.0 may result in star activity.

Reagents supplied: 10x M and 10x K buffer

Unit substrate: pBR322 DNA.

Unit calculation assay conditions: 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 50 units of Nae I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of pBR322 at 37^oC. After 10-fold overdigestion with Nae I, greater than 80% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Note: Nae I exhibits site preferences. pBR322 contains four Nae I recognition sequences. Two of these sites are readily cleaved, one is cleaved moderately slowly, and the fourth is cleaved 50-fold more slowly.

Reagents supplied: 10x L and 10x K buffer

BseBI is a restriction enzyme purified from Bacillus stearothermophilus.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH @ 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 60°C.

Absence of contaminants:  500 units of BseBI do not produce any unspecific  cleavage products after 16 hrs incubation with 1 μg of λ DNA at 60^oC. After ten-fold overdigestion with BseBI, less than 50% of the DNA fragments can be ligated.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Note: BseBI-cut DNA is difficult to ligate with T4 DNA Ligase. Ligation is enhanced in the presence of 15% PEG4000.

Reagents supplied: 10x M buffer

BseA I

(BspMII isoschizomer)

BseAI is a restriction enzyme purified from Bacillus stearothermophilus.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 10 mM Tris-HCl (pH 8.0 @ 25°C), 5 mM MgCl₂, 1 mM dithiothreitol, 0.02% Triton X-100, 100 μg/ml BSA. Incubate at 55^oC.

Absence of contaminants: 400 units of BseAI do not produce any unspecific  cleavage products after 16 hrs incubation with 1 μg of λ DNA at 55^oC. After 100-fold overdigestion with BseAI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM  Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Reference: Thanos, D., Scarpelis, G., Papamatheakis, J. and Bouriotis, V. (1989). Nucleic Acids Res. 17, 8881.

Reagents supplied: 10x U_BseAI and 10x K buffer

MspC I

(Afl II isoschizomer)

MspCI is a restriction enzyme purified from Micrococcus species.

Unit substrate: Lambda DNA (Hind III digest).

Unit calculation assay conditions: 150 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 80 units of MspCI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/Hind III digest at 37^oC. After 10-fold overdigestion with MspCI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reference: Rina, M., Tzanodaskalaki, M., Karagouni, A., Pagomenou, M. and Bouriotis, V. (1992). Nucleic Acids Res., 20, 1806.

Reagents supplied: 10x SH and 10x K buffer

BglII is a restriction enzyme purified from Bacillus globigii lacking BglI.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 150 units of BglII do not produce any unspecific  cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37^oC. After 50-fold overdigestion with BglII, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Reagents supplied: 10x H and 10x K buffer

Unit substrate: Lambda DNA (dam^-).

Unit calculation assay conditions: 100 mM KCl, 10 mM Tris-HCl (pH 8.0 @ 25°C), 10 mM MgCl₂, 1mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 20 units of Mbo I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA (dam^–) at 37^oC. After 10-fold overdigestion with Mbo I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes..

Methylation Sensitivity:

dam methylation: Blocked

dcm methylation: Not sensitive

CpG methylation: Impaired by overlapping

Reagents supplied: 10x U_MboI and 10x K buffer

Unit substrate: Lambda DNA (EcoRI digest).

Unit calculation assay conditions: 10 mM Tris-HCl (pH 7.0 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 0.01% Triton X-100, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 30 units of Kpn I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of lambda DNA/EcoR I digest at 37^oC. After 10-fold overdigestion with KpnI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5% or pH >8.0 may result in star activity.

Reagents supplied: 10x U_KpnI and 10x K buffer

BglI is a restriction enzyme purified from Bacillus globigii lacking BglII.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM KCl, 20 mM Tris-HCl (pH 8.5 @ 25°C), 10 mM MgCl₂, 0.04% Triton X-100, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 100 units of BglI do not produce any unspecific  cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37^oC. After 50-fold overdigestion with BglI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 300 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

Reagents supplied: 10x U_BglI and 10x K buffer

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 50 units of Hpa I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 10-fold overdigestion with Hpa I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: No.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

Star activity: Conditions of high enzyme concentration or glycerol concen-tration >5%, may result in star activity.

Reagents supplied: 10x A and 10x K buffer

HinfI is a restriction enzyme purified from a recombinant E.coli strain.

Unit substrate: Lambda DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin and DNA. Incubate at 37^oC.

Absence of contaminants: 200 units of Hinf I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC. After 100-fold overdigestion with Hinf I, greater than 90% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml bovine serum albumin and 50% glycerol. Store at -20^oC.

Heat inactivation: 80^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping