Aladdin HindIII is a genetically engineered recombinant fast restriction endonuclease designed for rapid digestion of plasmid DNA, PCR products, and genomic DNA. The enzyme exhibits excellent activity in either the universal 10× Buffer or 10× Color Buffer, enabling complete digestion within 5–15 minutes.
The 10× Color Buffer contains red and yellow tracking dyes, allowing the digestion products to be loaded directly onto agarose gels for electrophoresis. In a 1% agarose gel, the red dye migrates at a rate comparable to that of a 2500 bp double-stranded DNA fragment, while the yellow dye migrates at a rate comparable to that of a 10 bp double-stranded DNA fragment.
Contents & Storage
Component No. | Component Name | Appearance | 500 rxns | Storage |
H1540147-A | HindIII | Liquid | 500 μL | -20 ℃ |
H1540147-B | 10×Buffer | Liquid | 2×1 mL | -20 ℃ |
H1540147-C | 10×Color Buffer | Liquid | 2×1 mL | -20 ℃ |
Recommended Reaction Conditions
1× Buffer;
Incubate at 37 °C;
Prepare the reaction mixture according to the "Rapid DNA Digestion Protocol."
Inactivation Conditions
Incubate at 80 °C for 20 min to inactivate the enzyme.
Quality Control
1. Functional Activity Assay
At 37 °C, 1 μL of HindIII completely digests 1 μg of λDNA within 15 min in a 20 μL reaction mixture containing the universal buffer.
2. Prolonged Incubation Assay
At 37 °C, 1 μL of HindIII is incubated with 1 μg of λDNA for 3 h in a 20 μL reaction mixture containing the universal buffer. No nonspecific substrate degradation caused by contaminating nucleases or star activity is detected. Prolonged digestion beyond this incubation period may result in star activity.
3. Digestion-Ligation-Redigestion Assay
DNA substrates are digested with a 10-fold excess of HindIII at 37 °C. After purification, more than 95% of the digested DNA fragments can be religated using T4 DNA Ligase (Fast) at 22 °C. Following recovery of the ligation products, more than 95% can be cleaved again using the same restriction endonuclease.
4. Nonspecific Endonuclease Activity Assay
At 37 °C, 1 μL of HindIII is incubated with 1 μg of supercoiled plasmid DNA for 4 h in a 20 μL reaction mixture containing the universal buffer. Subsequent agarose gel electrophoresis analysis shows that less than 10% of the plasmid DNA is converted into nicked or linear forms.
5. Blue-White Screening Assay
A specific vector containing the lacZα gene and a single restriction site within this gene is digested with 1 μL of HindIII. Following religation, the DNA products are transformed into competent Escherichia coli cells and plated onto LB agar containing X-gal, IPTG, and the appropriate antibiotic.
Successful religation restores functional β-galactosidase expression, resulting in blue colonies. In contrast, DNA fragments that fail to religate due to restriction-end degradation or other factors produce white colonies. For HindIII, the proportion of white colonies should be less than 1%.
Instructions for Use
1. Rapid DNA Digestion Protocol
① Prepare the reaction mixture on ice in the recommended order of reagent addition as follows:
Component | Plasmid DNA | PCR Product | Genomic DNA |
ddH₂O | 15 μL | 16 μL | 30 μL |
10× Buffer or 10× Color Buffer | 2 μL | 3 μLᵃ | 5 μL |
Substrate DNA | 2 μL (up to 1 μg) | 10 μL (approximately 0.2 μg) | 10 μL (5 μg) |
HindIII | 1 μL | 1 μL | 5 μL |
Total Volume | 20 μL | 30 μL | 50 μL |
Note a: This reaction system is suitable for the digestion of purified PCR products. Unpurified PCR products exhibit a certain degree of ionic strength; therefore, the volume of 10× Buffer may be reduced to 2 μL as appropriate. However, residual DNA polymerase may possess exonuclease activity that can affect the digestion products. Therefore, if subsequent cloning or related procedures are required, purification of PCR products before restriction digestion is recommended.
② Mix gently by pipetting up and down or tapping the tube (do not vortex), then briefly centrifuge to collect droplets from the tube walls.
③ Incubate at 37 °C for 15 min (plasmid DNA), 15–30 min (PCR products), or 30–60 min (genomic DNA).
④ Incubate at 80 °C for 20 min to inactivate the enzyme and terminate the reaction (optional).
⑤ If 10× Color Buffer is used for the digestion reaction, the resulting products can be loaded directly onto an agarose gel for electrophoresis.
2. Double or Multiple Restriction Digestion
① Use 1 μL of each fast restriction endonuclease and scale up the reaction volume as necessary.
② The combined volume of all fast restriction endonucleases must not exceed one-tenth of the total reaction volume.
③ If the restriction endonucleases have different optimal reaction temperatures, initiate digestion with the enzyme requiring the lower optimal temperature. Subsequently, add the enzyme requiring the higher optimal temperature and perform the digestion at its optimal reaction temperature.
3. Scaled-Up Reaction Systems for Plasmid DNA
Component | 1 μg DNA | 2 μg DNA | 3 μg DNA | 4 μg DNA | 5 μg DNA |
DNA | 1 μg | 2 μg | 3 μg | 4 μg | 5 μg |
HindIII | 1 μL | 2 μL | 3 μL | 4 μL | 5 μL |
10× Buffer or 10× Color Buffer | 2 μL | 2 μL | 3 μL | 4 μL | 5 μL |
Total Volume | 20 μL | 20 μL | 30 μL | 40 μL | 50 μL |
Note: If the total reaction volume exceeds 20 μL, the incubation time should be extended appropriately. The use of a water bath, dry block heater, or sand bath is recommended whenever possible.
Number of Restriction Sites in Different DNA Substrates
λDNA | ΦX174 | pBR322 | pUC57 | pUC18/19 | SV40 | M13mp18/19 | Adeno2 |
6 | 0 | 1 | 1 | 1 | 6 | 1 | 12 |
Effects of DNA Methylation
Dam | Dcm | CpG | EcoKI | EcoBI |
No Effect | No Effect | No Effect | No Effect | Cleavage Inhibited |
