Endonucleases Non-Specific, HL-SAN

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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.

Recommended applications:

Complete removal of DNA 

Viscosity reduction

Key advantages with this Salt Active Nuclease:

• Non specific endonuclease
• Optimum activity at high salt concentration (0.5 M NaCl)
• Active at low temperatures (20% at 6ºC)
• Easily inactivated
• Broad pH range
• Temperature stable

Figures

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.

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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.

Overview

• Efficient depletion of bovine exosomes from Fetal Bovine Serum
• Deplete exosome-sized vesicles from versatile FBS volumes
• No protease treatment required
• No time-consuming ultracentrifugation
• No precipitation reagents required
• No overnight incubation required
• Exosome depletion confirmed by reduction of bovine miRNAs below detectable levels
• The depleted FBS provides the same cellular growth rates as the standard FBS
• Purification is based on Norgen’s proprietary Silicon Carbide resin matrix

Norgen’s FBS Exosome Depletion Kits provides a quick and easy protocol for the depletion of bovine exosomes from FBS prior to using it as a growth supplement in your culture medium. The FBS recovered from the depletion process is exosome-depleted and does not contain any quantifiable bovine miRNAs. Moreover, the exosome-depleted FBS will support the growth of your cells of interest similar to the non-depleted FBS. Norgen’s kits allow for the processing of different FBS volumes. The depletion is based on Norgen’s proprietary resin. These kits provide a clear advantage over other available kits in that they do not require ultracentrifugation, any special instrumentation, precipitation reagents or any protease treatments. More importantly, the depletion process is an inexpensive method for exosome depletion from FBS, as compared to the expensive current ready-to-use exosome-depleted media available on the market.

Background

Most culture medium used for the growth and propagation of cells in culture require the addition of fetal bovine serum (FBS) as a growth supplement to media. FBS is obtained from bovine (cow) serum, and therefore contains large quantities of bovine exosome vesicles. These exosomes may interfere with some types of studies, or may lead to unreliable results when studying the exosomes shed from your cells of interest in normal culture conditions. Therefore, the use of exosome-depleted FBS is highly recommended for many types of studies.

FBS Exosome Depletion Kit (Slurry Format)

For FBS volumes ranging from 140 mL to 280 mL.

FBS Exosome Depletion Kit (Column Format)

For FBS volumes ranging from 120 mL to 240 mL.

Details

Supporting Data

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Kit Specifications
Sample Type	Fetal Bovine Serum
Sample Volume Range	Up to 140 mL (FBS Exosome Depletion Kit I (Slurry Format) Up to 280 mL (FBS Exosome Depletion Kit II (Slurry Format)
Depletion	Deplete exosome-sized vesicle
Bovine miRNA	No detectable bovine miRNA
Time to Complete 6 Purifications	40 minutes

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Component	Cat. 61100 (6 preps)	Cat. 61400 (12 preps)	Cat. 61200 (6 preps)	Cat. 61300 (12 preps)
ExoC Buffer	2 x 1.5 mL	8 mL	2 x 1.5 mL	8 mL
Slurry E	12.5 mL	2 x 12.5 mL	–	–
Maxi Spin Column	–	–	6	12
Product Insert	1	1	1	1

Introduction

This product is suitable for rapid RNA extraction from tissue , cells, and other clinical samples. RNA can be used directly for RT-PCR, quantitative RT-PCR and so on.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from tissue, cell
Applications	RT-PCR, cDNA synthesis, second generation sequencing
Purification method	Polydisperse magnetic beads
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Adaptive instrument	Nucleic acid extractor, pipetting workstation
Sample type	Tissues, cells, lymphocytes and other clinical sample
Sample amount	Cells grown in suspension:3~5 x 106Animal tissue: 10~20mgPlant tissue: ≤100mg

Principle

The Kit combines the speed and efficiency of silica-based technology with the convenient handling of magnetic particles for purification of total RNA. Samples are lysed and RNA is purified from lysates in one step through its binding to the silica surface of the particles in the presence of a chaotropic salt. The particles are separated from the lysates using a magnet and DNA is removed by treatment with RNase-free DNase I. The magnetic particles are efficiently washed, and RNA is eluted in RNase-free water

Advantages

• High purity – OD260 / 280 :1.9-2.0, OD 260 / 230 :1.5-2.0
• Economy – less than 50% of the price of Qiagen and other imported products
• Automatic – extraction without manual participation, saving time and effort
• Universal – suitable for various clinical samples

Kit Contents

Contents	IVD3020
Purification Times	200 Preps
MagPure RNA Particles	7 ml
DNase I	4 x 600 µl
DNase Buffer	80 ml
RTL Lysis Buffer	150 ml
Buffer MCB*	75 ml
Buffer MW1*	110 ml
Buffer MW2*	50 ml
RNase Free Water	60 ml

Cat.No	Reagent	IVD3020-F-96
DNase Buffer		60 ml
DNase I		2 x 600 μl
RTL Lysis Buffer		80 ml
Buffer MCB		18 ml
96-Tip		1
Sample plate (DW Plate)	500μl Buffer MCB	1
Wash 1 Plate (DW Plate)	700μl Buffer MW130μl MagPure RNA Partilces	1
DNase Plate	Empty
Wash 2 Plate (DW Plate)	700μl Buffer MW1	1
Wash 3 Plate (DW Plate)	900μl Buffer MW2	1
Elution plate (DW Plate)	80μl RNase Free Water	1

Cat.No	Reagent	IVD3020-TL-06
Purification times		96 Preps
DNase I		2 x 600 μl
DNase Buffer		60 ml
RTL Lysis Buffer		60 ml
Buffer MCB		40 ml
96-Tip		12 PCS
2.0ml V-bottom plate	Row 1/7：500μl Buffer MCBRow 2/8：500μl Buffer MW1Row 3/9：emptyRow 4/10：30μl Magpure RNA Particles500μl Buffer MW2 Row 5/11：900μl Buffer MW2 Row 6/12：80μl RNase Free Water	6

Storage and Stability

MagPure RNA Particles should be stored at 2–8°C upon arrival. DNase I should be stored at -20°C. However, short-term storage (DNase I up to 1 weeks, MagPure RNA Particles up to 8 weeks) at roomtemperature (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 these conditions.

This product is suitable for rapid RNA extraction from tissue , cells, and other clinical samples. RNA can be used directly for RT-PCR, quantitative RT-PCR and so on.