Endonucleases Non-Specific, HL-SAN
HL-SAN efficiently removes nucleic acids from buffers typically used in protein purification. Due to its high salt tolerance, it is the obvious choice for host-cell DNA removal in settings where salt is added to reduce aggregation. Especially efficient for removing nucleic acids from proteins with high affinity for DNA and RNA. Proven performance during lysis and early stages of protein purification processes, as well as high-salt eluates. Cold-adapted enzyme with excellent performance also at ambient temperatures and during over-night digestion at 4°C.
Figure 1. Optimum activity in solutions with high salinity
HL-SAN has optimum activity at ∼0.5 M NaCl, but operates at a broad range of [NaCl] and [KCl]. The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2 with varying [NaCl] or [KCl]. The maximum activity was set to 100%.
Figure 2. Temperature and activity
HL-SAN has optimum activity at ~35°C, but works over a broad temperature range (20% activity at 10°C and 50°C). The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5 containing 5 mM MgCl2 and 0.5 M NaCl.
Fig 3. The effect of MgCl2 and MnCl2 concentration on the HL-SAN activity.
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 0.5 M NaCl and with varying concentrations of MgCl2 or MnCl2. The activity of the sample containing 5 mM MgCl2 was set to 100%.
Figure 4. HL-SAN activity vs pH/[NaCl]
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer with different pHs and different concentrations of NaCl. All buffers contained 5 mM MgCl2. The nature of the buffer was pH-dependent, but generally the NaCl-optimum was the same in all buffers/pHs. The exception was etanolaminbuffer at pH 9 and pH 9.5 in which the NaCl-optimum was shifted to the left (not shown).
Figure 5. Buffer composition affects substrate preference
Without NaCl, the specificity towards ssDNA and dsDNA is similar. At 0.5 M NaCl, the activity towards dsDNA increases, while the activity towards ssDNA is unaffected.
Figure 6. HL-SAN digests ssDNA to ~5-13 nt, and dsDNA to ~5-7 nt
The size of the end products from ssDNA varies from ~5-13 nt, while dsDNA is digested to around ~5-7 nt. The size of the end products seems to depend on the DNA sequence. Substrates 1 and 2 were ssDNA with different sequences and substrates 3 and 4 were dsDNA with similar sequences but with a FAM-label at different ends. Substrate 5 was dsDNA with the same sequence as substrate 3 and 4 but with a FAM-label at both ends.
Figure 7. HL-SAN activity decreases with increasing concentrations of glycerol
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with increasing concentrations of glycerol. The activity of the control not containing glycerol was set to 100%.
Figure 8. The activity of HL-SAN at different concentrations of imidazole
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with varying concentrations of imidazole. The activity of the control not containing imidazole was set to 100%.
HL-SAN efficiently removes nucleic acids from buffers typically used in protein purification. Due to its high salt tolerance, it is the obvious choice for host-cell DNA removal in settings where salt is added to reduce aggregation. Especially efficient for removing nucleic acids from proteins with high affinity for DNA and RNA. Proven performance during lysis and early stages of protein purification processes, as well as high-salt eluates. Cold-adapted enzyme with excellent performance also at ambient temperatures and during over-night digestion at 4°C.
Hipure viral RNA kit is suitable for purifying viral RNA from samples such as cell-free body fluid or culture medium. The kit is based on silica gel column purification technology. It requires no toxic phenol chloroform extraction and time-consuming alcohol precipitation in the extraction. The whole extraction process takes only 25 minutes. The kit is suitable for extracting viral RNA from 1-140µl cell-free fluid samples such as serum, plasma, cell-free body fluid or culture medium. The product has successfully extracted hepatitis B A/C, hepatitis C RNA, SARS and HIV. The obtained RNA can be directly used for RT-PCR, Northern hybridization and virus detection.
Specifications
| Features | Specifications |
| Main Functions | Extract viral RNA from 140μl cell-free samples |
| Applications | RT-PCR, Northern hybridization, and various virus detection |
| Purification method | Mini spin column |
| Purification technology | Silica technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | Cell-free body fluid or culture medium |
| Sample amount | 140μl |
| Elution volume | ≥15μ |
| Time per run | ≤25 minutes |
| Liquid carrying volume per column | 800μl |
| Binding yield of column | 100μg |
Hipure silica gel column is based on glass fiber filter membrane with high binding force. Under the condition of high concentration of ionizing agent (such as guanidine hydrochloride or guanidine isothiocyanate), the filter membrane can adsorb nucleic acid through hydrogen bond and electrostatic, while protein and other impurities are not adsorbed and removed. The filter membrane adsorbed with nucleic acid is washed to remove the residual protein and salt. Finally, the nucleic acid adsorbed on the filter membrane can be washed out with low salt buffer (such as buffer TE) or water. The obtained nucleic acid has high purity and can be directly used in various downstream experiments.
The sample is homogenized and lysed in the lysate, and RNA is released into the lysate. The high concentration of guanidine isothiocyanate contained in the lysate denatured and inactivated endogenous or exogenous RNase, while RNA is protected from degradation. The lysate is centrifuged to remove insoluble impurities. After adding ethanol to adjust the binding conditions, it is transferred to the column for filtration. RNA is adsorbed on the membrane of the column, while protein is removed without adsorption. The column is washed with buffer VHB to remove protein and other impurities, washed with buffer RW2 to remove salt. Finally the RNA is eluted by RNase free water. The eluted RNA can be directly used in RT-PCR, Northern blot, poly-A purification, in vitro translation, etc.
Advantages
Kit Contents
| Contents | R417102 | R417103 |
| Purification Times | 50 Preps | 250 Preps |
| HiPure Viral Micro Columns | 50 | 250 |
| 2ml Collection Tubes | 100 | 500 |
| Buffer VRL | 50 ml | 200 ml |
| Carrier RNA | 310 µg | 3 x 310 µg |
| Buffer VHB* | 13 ml | 110 ml |
| Buffer RW2* | 20 ml | 2 x 50 ml |
| Nuclease Free Water | 10 ml | 30 ml |
Storage and Stability
Carrier RNA should be stored at 2–8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15–25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15–25°C) and are stable for at least 18 months under theseconditions
Hipure viral RNA kit is suitable for purifying viral RNA from samples such as cell-free body fluid or culture medium. The kit is based on silica gel column purification technology. It requires no toxic phenol chloroform extraction and time-consuming alcohol precipitation in the extraction. The whole extraction process takes only 25 minutes. The kit is suitable for extracting viral RNA from 1-140µl cell-free fluid samples such as serum, plasma, cell-free body fluid or culture medium. The product has successfully extracted hepatitis B A/C, hepatitis C RNA, SARS and HIV. The obtained RNA can be directly used for RT-PCR, Northern hybridization and virus detection.
Hipure Stool RNA Kit is specially designed for stool RNA extraction. This kit is suitable for extracting high-purity microbial or host cell RNA from ≤0.1g stool samples. The kit adopts silica gel column purification technology and original solution system, which can effectively remove humic acid and other inhibitory factors in stool samples. The purified RNA can be directly used in RT-PCR, Northern hybridization and other experiments.
Specifications
| Features | Specifications |
| Main Functions | Isolation total RNA from 100-150mg stool sample |
| Applications | RT-PCR, Northern hybridization and other experiments |
| Purification method | Mini spin column |
| Purification technology | Silica technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | Stool |
| Sample amount | 100-150 mg |
| Elution volume | ≥30μl |
| Time per run | ≤50 minutes |
| Liquid carrying volume per column | 100µg |
| Binding yield of column | 800µl |
The HiPure silica gel column uses a high binding ability glass fiber filter membrane as the substrate. Under the condition of high concentration of ionizing agent (such as Guanidinium chloride or guanidine isothiocyanate), the filter membrane can adsorb nucleic acid through hydrogen bonding and electrostatic and other physical factors, while protein or other impurities are not adsorbed and removed. The filter membrane that has adsorbed nucleic acids is washed to remove proteins and salts. Finally, low salt buffer solution (such as Buffer TE) or water can be used to wash out the nucleic acids adsorbed on the filter membrane. The obtained nucleic acid has high purity and can be directly used in various downstream experiments.
The stool samples are homogenized in the lysis solution, further lysed in a high-temperature water bath, and RNA is released into the lysis solution. Chloroform extraction removes genomic DNA and impurities, transfer the supernatant to an alcohol free binding solution, purify RNA through a column, and finally elute RNA with RNase Free Water. The purified RNA can be directly used for experiments such as PCR, Southern hybridization, and enzyme digestion.
Advantages
Kit Contents
| Contents | R418502 | R418503 |
| Purification Times | 50 Preps | 250 Preps |
| HiPure RNA Mini Columns | 50 | 250 |
| 2ml Collection Tubes | 50 | 250 |
| Glass Beads (0.1~0.6mm) | 30 g | 150 g |
| Buffer SPL | 30 ml | 140 ml |
| Buffer PHC | 30 ml | 140 ml |
| Buffer GRP | 60 ml | 250 ml |
| Buffer RW1 | 50 ml | 250 ml |
| Buffer RW2 * | 20 ml | 2 x 50 ml |
| RNase Free Water | 15 ml | 30 ml |
Storage and Stability
The kit components can be stored at room temperature (15–25°C) and are stable for 18 months under these conditions. At low temperatures, Buffer SPL may form precipitates, dissolve it by 55°C water bath. After receiving the product, Buffer PHC should be stored at 2-8°C.
Hipure Stool RNA Kit is specially designed for stool RNA extraction. This kit is suitable for extracting high-purity microbial or host cell RNA from ≤0.1g stool samples. The kit adopts silica gel column purification technology and original solution system, which can effectively remove humic acid and other inhibitory factors in stool samples. The purified RNA can be directly used in RT-PCR, Northern hybridization and other experiments.