Mal-amido-PEG4-alkyne is a short PEG linker featuring a maleimide and a terminal alkyne. Maleimide is a covalent ligand used for linking thiols such as those in cysteines residues in proteins, while terminal alkynes can be used in copper (I) click chemistry with azides on target molecules.
Mal-amido-PEG4-alkyne is a short PEG linker featuring a maleimide and a terminal alkyne. Maleimide is a covalent ligand used for linking thiols such as those in cysteines residues in proteins, while terminal alkynes can be used in copper (I) click chemistry with azides on target molecules.
The HiPure Total RNA Kit provides fast purification of high-quality RNA from cells, tissues, and yeast using silica-membrane spin columns with a binding capacity of 100μg RNA. There is no need for phenol/chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or isopropanol precipitation. RNA purified using the HiPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
Specifications
| Features | Specifications |
| Main Functions | Isolation total RNA (not include miRNA) from animal tissues, cells and simple plant tissues using one column |
| Applications | RT-PCR, qRT-PCR, Northern hybridization, second generation sequencing |
| Purification method | Mini spin column |
| Purification technology | Silica technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | Animal soft tissue, cultured cells, lymphocytes, simple plant tissue |
| Sample amount | Cells: ≤1 x 107 Animal tissue: 1-20 mgPlant leaves: 50-150 mg Yeast cells: 5 x 106 |
| Yield | 2-100μg |
| Elution volume | ≥50μl |
| Time per run | ≤15 minutes（1-24 samples） |
| Liquid carrying volume per column | 800µl |
| Binding yield of column | 100µg |
HiPure RNA technology simplifies total RNA isolation. Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the HiPure silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment. Pure, concentrated RNA is eluted in water.
Advantages
Kit Contents
| Contents | R401102 | R401103 |
| Purification Times | 50 Preps | 250 Preps |
| HiPure RNA Mini Columns | 50 | 250 |
| 2ml Collection Tubes | 50 | 250 |
| RTL Lysis Buffer | 50 ml | 200 ml |
| RNA Binding Buffer | 15 ml | 75 ml |
| Buffer RW1 | 50 ml | 200 ml |
| Buffer RW2* | 20 ml | 2 x 50 ml |
| RNase Free Water | 10 ml | 30 ml |
Storage and Stability
The Kit can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions.
The HiPure Total RNA Kit provides fast purification of high-quality RNA from cells, tissues, and yeast using silica-membrane spin columns with a binding capacity of 100μg RNA. There is no need for phenol/chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or isopropanol precipitation. RNA purified using the HiPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
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.
