Nucleic Acid Purification Kits

Nucleic acid purification kits are used to extract and purify nucleic acids (DNA or RNA) from your samples, including cells, tissues, etc. DNA or RNA is an organic molecule that store genetic information in the cells. To study them, we have to isolate and purify the nucleic acids from the unwanted component in the cells. But before that, let’s have a look at what are the nucleic acids.

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What are the nucleic acids?

Living organisms on this earth, either unicellular or multicellular organisms, contain two major types of nucleic acids. They are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Both of them made from nucleotides, organic molecules that contain five-carbon sugar backbones, phosphate groups and nitrogen bases.

Deoxyribonucleic Acid (DNA)

DNA is a polymer of 4 nucleotides, Adenine (A), Thymine (T), Cytosine (C) and Guanine (G), where A pair with T and C pair with G. They contain all the genetic code that is essential for development and function of every single cell. Besides, they are the fundamental molecules that make us different from each other!

Ribonucleic Acid (RNA)

Similar to DNA, RNA is also a polymer of 4 nucleotides, Adenine (A), Uracil (U), Cytosine (C) and Guanine (G). Instead of Thymine as in DNA, RNA contains Uracil (U), which also pair with Adenine (A).

Up to date, there are three main types of RNA:

1. Messenger RNA (mRNA) – its primary role is to encode the amino acid sequence of polypeptides, which eventually become part of a protein.
2. Ribosomal RNA (rRNA) – it is part of the ribosomes, which responsible for translation, in other words, synthesis of protein.
3. Transfer RNA (tRNA) – it helps to bring amino acid to the ribosome and decode the mRNA sequence into protein.

Why need to purify DNA and RNA from your samples?

DNA or RNA purifications are the fundamental steps for any downstream application in biotechnology and molecular biology study. Without purifying your samples, it may contain many contaminants that will lead to erroneous result in your research.

Hence, using DNA or RNA isolation kit can help us to:

1. Obtain high quality of DNA and RNA from your sample
2. Reduce the amounts of contaminants which may shorten the shelf life of your DNA and RNA

For different applications, we need to use different kinds of purification kits.

Monarch Plasmid Miniprep Kit

Plasmid is a small circular DNA molecule that physically separated from chromosomal DNA. They commonly found in bacterial cells, and sometimes in archaea and eukaryotic organisms. They can replicate independently and often carry genes that benefit the cells survival. One of the common genes is the antibiotic resistance gene.

Scientists usually used plasmid for transformation, transfection, DNA sequencing, restriction digestion and other enzymatic manipulations. In order to do so, the high quality of purified plasmid DNA is a must!

The Monarch Plasmid Miniprep Kit is a fast and reliable kit to purify the high quality of plasmid DNA. Here, it employs standard cell resuspension, alkaline lysis, and neutralization steps. To remove all the unwanted substances, like salts, proteins, RNA and other cellular components, NEB uses a unique washing buffer to do so, leaving low-volume elution of concentrated and highly pure DNA. Besides, NEB included colour indicators to ease users in monitoring the completion of extraction.

What do you need to know before using Monarch Plasmid Miniprep Kit?

Here, we show you the specification in the below table:

DescriptionSpecificationsCulture VolumeRecommended between 1 to 5 ml and not to exceed 15 OD unitsBinding CapacityUp to 20μgPlasmid SizeUp to 20kbTypical RecoveryUp to 20μg. Yield is affected by several factors, including plasmid copy number, host strain, culture volume, and growth conditionsElution VolumeElute in as little as 30μLPurityA260/280 and A260/230 ≥ 1.8Protocol TimeSpin and incubation times: 10.5 minsDownstream ApplicationTransformation, Transfection, DNA sequencing, Polymerase Chain Reaction (PCR), Labelling, Restriction Digestion and Other Enzymatic Manipulations, etc.

Advantages of using Monarch Plasmid Miniprep Kit

• Low elution volume: as little as 30μL
• Optimized column design to prevent buffer retention and salt carry-over – say no more to contamination!
• Faster protocols and less spin time – saving you valuable time
• Easily keep track of the certain steps by observing the colour changes of buffer
• No RNase needed before starting – save money
• Easily label columns – tab and frosted surface
• Buffers and columns can be purchased separately
• Go GREEN – significantly less plastic is used (compare to other kits) and recyclable packaging
• No hazardous materials fees – save money and save the environment!

Basic Steps for Plasmid Purification using Monarch Plasmid Miniprep Kit

**Before you start, add 4 volumes of ethanol to 1 volume of DNA wash buffer.

**All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Pelleting

Pellet your cells between 1-5ml of bacterial cell culture by centrifuge at g / rpm for 30 seconds. Carefully discard the supernatant.

Resuspend Pellet

Resuspend you pellet with 200μl Monarch Plasmid Resuspension Buffer (B1, Pink in colour). Vortex or pipet for thorough mixing until completely resuspended.

Tips: Resuspend cells completely to achieve complete lysis in the following steps.

Lyse Bacteria

Add 200μl of Monarch Plasmid Lysis Buffer (B2, Blue in colour) to the cell suspension. Mix gently by inverting the tubes 5-6 times until the colour changes to dark pink. Incubate for 1 min at room temperature to ensure complete lysis of the cells.

Tips: Do not vortex the solution as this may shear your DNA and the quality and yield of extracted DNA can be affected.

Neutralize Lysate

Add 400μl of Monarch Plasmid Neutralization Buffer (B3, Yellow in colour) to the solution. Gently mix the solution by inverting the tube 4-6 times. Ensure that you mix the solution until uniformly yellow and precipitate forms. Incubate for 2 mins at room temperature and centrifuged for 2-5 mins.

Tips: Longer spin times will result in a more compact pellet, which will lower the risk of clogging the column.

Binding DNA to Column

Transfer the supernatant which contains the plasmid DNA to the spin column and centrifuge for one min. The DNA is now bound to the column matrix. Remove the column and discard the flow-through.

Washing Steps

Insert column into the collection tube and add 200μl of Monarch Plasmid Wash Buffer 1. Centrifuge for 1 min. Optional: You may discard the flow-through. Add 400ul Monarch Plasmid Wash Buffer 2 and centrifuge for 1 min. Transfer the column to the clean centrifuge tube.

Tips: Ensure the column doesn’t touch flow-through during transfer.

Elution

Add minimum 30ul Monarch DNA Elution Buffer to the centre of the column matrix. Incubate the solution for 1 min at room temperature. Centrifuge 1 min and collect the flow-through and your purified plasmid DNA is ready for any of your downstream application.

Tips: Incubation less than 1 min can reduce the yield of DNA.

For video, please watch Protocol for Monarch Plasmid Miniprep Kit.

How to maximize yield when using Monarch Plasmid Miniprep Kits?

Here are some tips to maximize the yield of plasmid DNA.

• Ensure that the pellet is resuspended completely
  • Bacteria pellet that is not resuspended completely won’t be lysed efficiently and the yield of DNA after purification is lower.
• Ensure the solution is mixed completely by inverting the tubes gently during cell lysis.
• Make sure you add the elution buffer to the centre of the matrix without puncturing it. This will increase the efficiency of elution.
• Be sure to incubate the elution buffer in the column for a full minute before spinning.

Monarch PCR & DNA Cleanup Kit

After PCR or other enzymatic reactions, you might need to purify the DNA, for use in restriction digests, DNA sequencing, ligation or other enzymatic manipulations. With Monarch PCR & DNA Cleanup kit, you can easily and quickly purify the high quality of DNA.

This kit utilizes a bind/wash/elute workflow with minimal incubation and spin times. Besides DNA, the protocol can be modified to purify smaller DNA fragments, including oligonucleotides and ssDNA.

What do you need to know about Monarch PCR & DNA Cleanup Kit?

DescriptionSpecificationsDNA Sample Type

• DNA from PCR or other enzymatic reaction
• ssDNA or dsDNA oligonucleotides can also be purified with using Oligonucleotide Cleanup Protocol

Binding CapacityUp to 5μgDNA Size Range

• ~50 bp to 25 kb
• DNA ≥ 15 bp to 25 kb (dsDNA) and DNA ≥ 18 nt to 10 kb (ssDNA) can also be purified using the Oligonucleotide Cleanup Protocol

Typical Recovery

• DNA (50 bp to 10 kb): 70–90%
• DNA (11–23 kb): 50–70%
• ssDNA ≥ 18 nt and dsDNA ≥ 15 bp: 70-85%

Elution Volume≥ 6 μlPurityA260/280 > 1.8 and A260/230 > 1.8Protocol Time5 minutes of spin and incubation timeDownstream ApplicationLigation, restriction digestion, labelling and other enzymatic manipulations, library construction and DNA sequencing.

In what applications you should use Monarch PCR & DNA Cleanup Kit?

ApplicationExplanationPCR cleanupPurify your DNA by removing the polymerases, primers, detergent, etc. that used in PCR amplification.Enzymatic Reaction CleanupRemove the restriction enzymes and modifying enzymes (ligases, kinases, nucleases, etc) and allowing for effective desalting and concentration of your DNA sample.cDNA CleanupPurify your cDNA by removing RT nd polymerase as well as nucleotides.Labelling CleanupRemove unincorporated radiolabeled or fluorescently labelled nucleotides from your DNA.Plasmid CleanupPlasmid from unknown sources may contain inhibitors and unwanted contaminants. You can remove these unwanted substances using this kit.

Advantages of using Monarch PCR & DNA Cleanup kit

• Low elution volume: elute in as little as 6 μl
• Prevent buffer retention and salt carry – over with optimized column design – say no more to contamination!
• Save time with fast, user-friendly protocols
• No need to monitor pH, as NEB already optimized the buffer solution for you
• Protocol modification allows for ssDNA purification, oligonucleotide purification, and purification of other small DNA fragments
• Buffers and columns available separately – which means you can buy either buffers or columns only if you have finished one of them
• GO GREEN! – Significantly less plastic used when compared with other kits and responsibly-sourced and recyclable packaging

Monarch PCR and DNA Cleanup Kit Protocols

**Before you start, add 4 volumes of ethanol to 1 volume of DNA wash buffer.

**All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Dilution of Sample

Only use 20-100μL of your DNA sample and dilute with Monarch DNA Cleanup Binding Buffer according to your sample type:

DescriptionSpecificationDNA Sample TypeDNA from PCR or other enzymatic reaction
ssDNA or dsDNA oligonucleotides can also be purified with using Oligonucleotide Cleanup ProtocolBinding CapacityUp to 5μgDNA Size Range~50 bp to 25 kb
DNA ≥ 15 bp to 25 kb (dsDNA) and DNA ≥ 18 nt to 10 kb (ssDNA) can also be purified using the Oligonucleotide Cleanup ProtocolTypical RecoveryDNA (50 bp to 10 kb): 70–90%
DNA (11–23 kb): 50–70%
ssDNA ≥ 18 nt and dsDNA ≥ 15 bp: 70-85%Elution Volume≥ 6 μlPurityA260/280 > 1.8 and A260/230 > 1.8Protocol Time5 minutes of spin and incubation timeDownstream ApplicationLigation, restriction digestion, labelling and other enzymatic manipulations, library construction and DNA sequencing.

When do you need to use Monarch PCR & DNA Cleanup Kit?

ApplicationExplanationPCR cleanupPurify your DNA by removing the polymerases, primers, detergent, etc. that used in PCR amplification.Enzymatic Reaction CleanupRemove the restriction enzymes and modifying enzymes (ligases, kinases, nucleases, etc) and allowing for effective desalting and concentration of your DNA sample.cDNA CleanupPurify your cDNA by removing RT nd polymerase as well as nucleotides.Labelling CleanupRemove unincorporated radiolabeled or fluorescently labelled nucleotides from your DNA.Plasmid CleanupPlasmid from unknown sources may contain inhibitors and unwanted contaminants. You can remove these unwanted substances using this kit.

Advantages of Monarch PCR & DNA Cleanup kit

• Low elution volume: elute in as little as 6 μl
• Prevent buffer retention and salt carry-over with optimized column design – say no more to contamination!
• Save time with fast, user-friendly protocols – save your valuable time
• No need to monitor pH, as NEB already optimized the buffer solution for you
• Protocol modification allows for ssDNA purification, oligonucleotide purification, and purification of other small DNA fragments
• Buffers and columns available separately – which means you can buy either buffers or columns only if you have finished one of them
• GO GREEN! – Significantly less plastic used when compared with other kits and responsibly-sourced and recyclable packaging

Monarch PCR and DNA Cleanup Kit Protocols

**Before you start, add 4 volumes of ethanol to 1 volume of DNA wash buffer.

**All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Dilute Your Sample

Only use 20-100μL of your DNA sample and dilute with Monarch DNA Cleanup Binding Buffer according to your sample type:

Sample TypeRatio of Binding Buffer of SampleExampledsDNA > 2kb (plasmids, gDNA)2:μl:100μldsDNA < 2kb (some amplicons, fragments)5:μl:100μlssDNA (cDNA, M13)7:μl:100μl

Tips: If your initial DNA sample volume is less than 20μL, you need to top up with TE buffer to at least 20μL. However, if your initial DNA sample volume is more than 800μL, you have to load the sample incrementally to avoid overflowing column.

Bind Your DNA to Column

Load your sample to a column seeded to a collection tube and close the gap. Spin for one min and the DNA is bound to the column matrix. Discard the flow-through.

Wash Your DNA

Reinsert the column into the collection tube and add 200μL of DNA Wash Buffer. Spin for one min. Repeat the wash steps by adding 200μL of DNA wash buffer and spin again for one min.

Elute Your DNA

Transfer the column into a clean tube. Add a minimum of 6μL DNA Elution Buffer to the centre of the column and incubate for one min for maximizing yield. Spin for one min and collect the flow-through, your DNA is now ready for any downstream application.

Tips: When transferring the column, make sure that the tip doesn’t touch the flow-through to avoid contamination.

If you want to save time, you can reduce the binding and elution spins to 30 seconds.

For video, please watch Monarch PCR and DNA Cleanup Kits Protocol.

How to maximize DNA yield when using Monarch PCR and DNA Cleanup Kit?

• Add elution buffer to the centre of the column matrix for efficient elution.
• For DNA > 10kb, heat elution buffer to 50°C prior to adding to the column to improve yield.
• Sequential elution may increase yield but will result in lower final DNA concentration

Monarch DNA Gel Extraction Kit

After running DNA gel electrophoresis, we usually want to extract the DNA interest from the agarose gel. By using Monarch DNA Gel Extraction kit, you can rapidly and reliably get up to 5μg of purified, high-quality DNA from agarose gels.

Like other purification methods, Monarch DNA Gel Extraction kit also utilizes a bind/wash/elute method in purifying DNA. On top of that, the column in this kit ensures zero buffer retention and no carryover contaminants, allowing low sample elution volume (6μL).

To make the kit as user-friendly as possible, NEB has optimized the buffers. You do not need to monitor pH during purification of DNA. What’s more exciting is you do not need isopropanol to melt the agarose, saving you a step and time. After elution, the eluted DNA is ready for use in restriction digests, DNA sequencing, ligation and other enzymatic manipulation.

Specifications of Monarch DNA Gel Extraction kit

DescriptionSpecificationDNA Sample Typedouble-stranded DNA from agarose gelsBinding Capacityup to 5 μgDNA Size Range~50 bp to 25 kbTypical RecoveryDNA (50 bp to 10 kb): 70–90%
DNA (11–23 kb): 50–70%Elution Volume≥ 6 μlPurityA260/280 > 1.8Protocol Time10 minutes of spin and incubation timeCompatible Downstream ApplicationLigation, restriction digestion, labelling and other enzymatic manipulations, library construction and DNA sequencing.

Advantages of using Monarch DNA Gel Extraction Kit

• Elute in as little as 6μl
• Prevent buffer retention and salt carry-over with optimized column design
• Save time with fast, user-friendly protocols
• No need to monitor pH or add isopropanol
• Buffers and columns available separately
• Significantly less plastic used when compared with other kits
• Responsibly-sourced and recyclable packaging

Monarch DNA Gel Extraction Kit Protocol

**Before you start, add 4 volumes of ethanol to 1 volume of DNA wash buffer.

**All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Excise Gel Fragment

Visualize your DNA fragment of interest under UV light and excise from the gel.

Tips: You have to cut the agarose as close to the DNA as possible and limit UV exposure to prevent DNA damage.

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Dissolve Agarose

Transfer your excised band to 1.5ml microcentrifuge tube and weigh the gel. Add 4 volumes of Monarch Gel Dissolving buffer to the tube (eg, add 400μl of buffer to the 100μg of gels). Incubate your sample between 37 – 55°C for 5 – 10 mins to melt the agarose. Mixing periodically to ensure the agarose is completely dissolved.

Tips: Incomplete dissolving results in lower yield. This is because undissolved agarose still containing DNA and may also clog the column which reduces the elution efficiency.

Bind DNA to Column

Once the gel slice is dissolved, load your sample to the column. Centrifuge it for 1 min.

Wash the DNA

Reinsert the column into the collection tube and add 200μL of DNA Wash Buffer. Centrifuge for 1 min. Repeat the wash steps by adding 200μL of wash buffer and centrifuge for 1 min.

Elute DNA

Transfer the DNA into a clean microfuge tube. Add a minimum of 6μL of DNA elution buffer to the centre of the column. Incubate for 1 min. Centrifuge for 1 min and collect the flow through.

Tips: Make sure that the tip doesn’t contact the flow-through.

For video, please watch the Monarch DNA Gel Extraction Kit.

Tips for Optimizing DNA Quality and Yield

• Do not incubate gel slice above 60°C as this will damage the DNA.
• Add elution buffer to the centre of the column matrix for efficient elution.
• For fragments >8kb, add 1.5 volume water after dissolving agarose.
• For plasmids >10kb, heat the elution buffer to 50°C before use.
• Binding and elution spins can be reduced to 30 seconds.

Monarch RNA Cleanup Kit (10, 50 and 500µg)

You may need to purify your RNA before any application involves the use of purifying RNA. The Monarch RNA Cleanup Kit helps you to purify concentrated and high-quality RNA (>25nt) from enzymatic reactions like labelling, capping, in vitro transcription and DNase I treatment.

Like other purification kits, this kit purifies RNA through a bind/wash/elute steps, with the advantage of minimal incubation and spin times. The eluted RNA is ready for a wide variety of applications, such as RT-PCR, RNA labelling and RNA Library Prep for Next Generation Sequencing (NGS). If you need to purify the smaller RNA fragments, you can modify the protocol to do so.

 There are three types of Monarch RNA Cleanup Kit, each of them comes with different binding capacity, 10ug, 50ug and 500ug.

Specification of Monarch RNA Cleanup Kit

DescriptionSpecificationRNA Sample TypeCleanup and concentration of RNA from enzymatic reactions (labeling, capping, in vitro transcription reactions, DNase I treatment)Binding Capacity10µgRNA Size Range≥ 25 nt ( ≥ 15 nt with modified protocol)Typical Recovery70–100%Elution Volume6–20 µlPurityA260/280 > 1.8 and A260/230 > 1.8Protocol Time5 minutes of spin and incubation timeCompatible Downstream ApplicationRT-PCR, Small RNA library prep for NGS, RNA Library Prep for NGS

Application of Monarch RNA Cleanup Kit

ApplicationExplanationRNA Cleanup and Concentration (including from the TRIzol aqueous phase)RNA purified by other methods can be further purifiedEnzymatic Reaction CleanupEnzymes such as RNA polymerases, DNase I, Proteinase K and phosphatases are removed allowing efficient desaltingIn vitro Transcription CleanupEnzymes and excess NTPs are removed to yield highly pure synthesized RNARNA Gel ExtractionPurification of RNA from agarose gelsRNA FractionationFractionation of RNA into small and large RNA pools

Advantages of Using Monarch RNA Cleanup Kit

• Ideal for cleanup and concentration of RNA after enzymatic treatments including DNase I, Proteinase K, labelling, capping or in vitro transcription (IVT)
• Efficiently purify RNA ≥ 25 nt (a simple modification enables purification of RNA ≥ 15 nt)
• Elute in ≥ 20 µl for concentrated RNA
• 70-100% RNA recovery
• Can be used to purify RNA from the aqueous phase following TRIzol® or similar extractions
• Simplified workflow with a single wash buffer
• Unique column design prevents buffer carryover and elution of silica particulates
• Columns and buffers are available separately
• Purified RNA is ready for use in a wide variety of downstream applications, including transfection

How to purify using Monarch RNA Cleanup Kit

** Add 4 volumes of ethanol to wash buffer

**All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Prepare your sample

Recommended starting sample volume: 50µL. If your sample volume is lower, you can adjust the volume with Nuclease-free Water. However, if your sample volume is large, you have to scale buffer volume accordingly.

Add binding buffer

Add 2 volumes of RNA Cleanup Binding Buffer to your sample.
For RNA ≥ 25 nucleotides, add 1 volume of ethanol.
For RNA ≥ 15 nucleotides, add 2 volumes of ethanol.

Tips: Do not VORTEX for mixing. Mix well by pipetting up and down or invert the tube gently.

Load sample onto the column

After you load your sample to the column, spin at xg for 1 min and discard the flow through. If your sample more than 900µL, repeat the steps as necessary as the column can only hold 900µL at a time.

Wash column

Reinsert the column into the collection tube. Add 500µL RNA Cleanup Wash Buffer and spin for 1 min, then discard the flow-through.

Repeat the column wash

Add 500µL RNA Cleanup Wash Buffer and spin for 1 min, then discard the flow-through.

Elution

Transfer the column to RNase-free 1.5ml microcentrifuge tube. Be careful of the tips of the column do not contact with the flow-through to prevent the traces of salts and ethanol are not carried to the next steps. If the tips contact with the flow-through, respin for 1 min.

After centrifuging, add different volume of RNase-free water to the column and incubate for different incubation time according to the binding capacities of Monarch RNA Cleanup Kit in the table below.

Volume of RNase-free water and incubation time for different binding capacities of Monarch RNA Cleanup kitBinding capacities of Monarch RNA Cleanup Kits (µg)Volume of RNase-free Water* (µL)Incubation Time Spin Time (min)106-20n/a15020-100n/a**50-1005 mins (room temp)1

* Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.
** A second elution can be performed to maximize RNA recovery.

After the addition of RNase free water (and incubation for T), centrifuge to elute RNA. The eluted RNA can be used immediately or stored at -70°C.

For video, please watch Monarch RNA Cleanup Kits Protocol.

Tips for using Monarch RNA Cleanup Kits

• Before loading the sample to the column, be sure to add RNA Cleanup Binding Buffer first, then add ethanol. The binding buffer serves to inactivate enzymatic activities. Hence, preserving samples integrity. Addition of ethanol ensures that the DNA bind to the column.
• Do not add more than 900uL to the column at one time as this will exceed the column reservoir capacity.
• Ensure the tip of the column does not touch the collection tube after washing.
• During elution, load the RNAse free water to the centre of the column matrix.
• Recovery of small RNA, <45nt is affected by the degree of secondary structure. If the recovery of small RNA is lower than expected, you can dilute the samples with 2 volumes of ethanol, instead of 1 volumes, in the earlier steps of the protocol. The additional ethanol disrupts the secondary structure and enhances the binding to the column. As a result, the ethanol shifted the cutoff size of RNA from 25nt to 15nt.
• To save time, you can reduce the spin time for binding and elution to 30 seconds, instead of 1 min.

Monarch Genomic DNA Purification Kit

To study the genomic DNA from living things, we first need to extract and purify the DNA from the biological samples. In Monarch Genomic DNA purification kit, it contains a comprehensive solution for cell lysis, RNA removal, and purification of intact genomic DNA. This kit helps you to extract genomic DNA from a wide variety of samples, such as cultured cell, blood and mammalian tissues as well as tough to lyse sample, including bacteria and yeast. If you want to extract and purify gDNA from the clinically-relevant sample, you may consider Monarch Genomic DNA Purification kit as your option.

Purified gDNA using this kit has a high DIN score and minimal RNA contamination. And the best thing is, it is ready for any downstream application. For example, library preparation for NGS, PCR and qPCR. On top of that, it is an excellent choice for upstream of long-read sequencing platforms as the purified gDNA has a peak size of >50kb.

What you should know before using Monarch Genomic DNA Purification Kit

DescriptionSpecificationInputCultured mammalian cells: up to 5 x 106 cells
Mammalian whole blood: 100 µl
Tissue: up to 25 mg, depending on tissue type
Bacteria: up to 2 x 109
Yeast: up to 5 x 107
Saliva: up to 500 µl
Buccal swabs
Genomic DNA requiring cleanupBinding Capacity30µg genomic DNAGenomic DNA SizePeak size > 50 kb for most sample types; may be lower for saliva and buccal swabsRNA ContentD< 1% (with included RNase A treatment)Elution Volume≥35 µl, but 100 µl is recommendedPurityA260/280 ≥ 1.8
A260/230 ≥ 2.0

Validated Sample Types

MouseTail
Ear
Liver
Kidney
SpleenHeart
Lung
Brain
Muscle
BloodRatLiver
Brain
Muscle–OthersDeer Muscle
Human Blood
Rabbit Blood
Pig Blood
Guinea Pig Blood
Cow Blood
Horse Blood
Dog Blood
Chicken Blood
HeLa CellsHEK293 Cells
NIH3T3 Cells
E. coli
Rhodobacter sp.
B. cereus
T. kodakarensis
S. cerevisiae
Saliva
Buccal swab

Advantages of using Monarch Genomic DNA Purification Kits

• For use with a broad range of sample types, such as cells, blood, and tissues (including fatty and fibrous tissues)
• Also works with tough to lyse samples (e.g., bacteria, yeast)
• Suitable for clinically-relevant samples (e.g., saliva, cheek)
• Obtain excellent yields of highly-pure DNA
• Extremely low residual RNA contamination (typically <1%)
• Isolates high molecular weight gDNA (peak size typically ≥ 50 kb)
• Use DNA directly in downstream applications, including PCR, qPCR and NGS
• Fast, user-friendly protocols with short lysis times and minimal hands-on time
• Can also be used to clean up previously extracted gDNA
• Includes RNase A
• Kit components available separately

Monarch Genomic DNA Purification Kit Protocols

Basically, the Genomic DNA Purification consists of two stages:

1. Sample lysis
2. Genomic DNA Binding and Elution

The protocols would be different for different sample types. You can check the details protocols below:

• Cells
• Tissues
• Blood
• Gram Negative Bacteria
• Gram Positive Bacteria and Archae
• Yeast
• Buccal Swab
• Saliva

Tips for Optimal Results

Tips on optimizing gDNA yields and quality

• Place the column in the centrifuge in a consistent orientation to optimize yield and purity. This ensures that the liquid takes the same path in each spin.
• Preheat the elution buffer to 60°C. This helps to increase the efficiency of elution and increase the yield.
• If more concentrated DNA is desired, reduced the elution volume from 100 to 35uL. However, this will lead to a reduction of yield for about 20-25%.
• Apply your sample only to the membrane and avoid upper column area.
• Do not transfer foam that formed during lysis.
• Avoid touching the column reservoir with pipette tips.
• Bind your DNA at 2 speeds to maximize binding and purity, first at g and latter at maximum speed. Spinning at the low speed gives DNA more opportunity to bind to the matrix and spinning at the maximum speed help to remove the remaining salts and proteins.
• Invert the column with wash buffer to optimize removal of salts.
• Confirm the heating block temperature is 60°C. If the temperature is above 60°C, the DNA may be partially denatured.

Tips for efficient lysis of tissue sample

• Use the recommended input amount of sample type according to the Monarch DNA Inputs, as using excess sample will clog the column and results in lower yield and poor DNA quality.
• Cut the tissue into small pieces to increase the surface area for enzymatic digestion.
• Ensure no tissue pieces are stuck at the bottom of the tube after lysis buffer and proteinase K are added.
• Carry out lysis in a thermal mixer to increase efficiency. If time is not limiting, extend the lysis time up to 3 more hours to obtain higher yields and lower RNA content.
• If using heat block, ensure to vortex sample every 20-30 mins to speed up lysis.
• To ensure optimal RNA digestion, add RNase A at the end of lysis to prevent degradation of the RNase A by the Proteinase K.
• When working with fibrous or fatty tissue, or tissues stored in RNAlater, centrifuge lysate or 3 mins at max speed. As if these fibres are not removed prior to adding to the column, they will stick to the membrane and prevent DNA binding, results in reducing yield and quality.

Conclusions

DNA and RNA purification refers to the process of isolating and extracting DNA or RNA molecules from biological samples. Purification is necessary to obtain high-quality and pure DNA or RNA for various downstream applications, such as sequencing, cloning, PCR, gene expression analysis, and other molecular biology techniques. DNA and RNA purification kits are essential tools used in molecular biology and genetic research to extract and purify nucleic acids (DNA or RNA) from biological samples such as cells, tissues, or fluids [1].

DNA and RNA purification kits are used for several important reasons:

1. Isolation of Pure Nucleic Acids: The primary purpose of these kits is to extract and purify DNA or RNA from biological samples. The kits isolate highly pure nucleic acids by removing contaminants such as proteins, lipids, salts, and other impurities. Pure nucleic acid samples are essential for accurate downstream analysis and experimentation.
2. High Yield: DNA and RNA purification kits are designed to maximize the yield of nucleic acids extracted from the samples. This ensures that researchers obtain sufficient amounts of DNA or RNA for specific applications, such as PCR, sequencing, cloning, or gene expression studies.
3. Consistency and Reproducibility: These kits provide standardized protocols and reagents, ensuring consistent and reproducible results across different experiments or laboratories. This is particularly important for research and clinical settings where reliable and comparable data are crucial.
4. Time and Cost Efficiency: DNA and RNA purification kits streamline the nucleic acid extraction process, reducing the time and effort required compared to manual extraction methods. The kits include optimized protocols and pre-packaged reagents, eliminating the need for researchers to source and prepare individual components. This saves time and minimizes the risk of errors. Additionally, the kits can be cost-effective in reducing reagent consumption and minimizing the potential for sample loss.

What are the Most Used Commercial Purification Test Kits?

Here are some examples of the most commonly used commercial DNA or RNA Purification Kits from our company, Virasens:

Viral Nucleic Acid Purification Kit VIRA01: Virasens Viral Nucleic Acid Purification kit enables the purification of viral nucleic acids (DNA and RNA) from samples such as serum, plasma, body fluids, and supernatant of virus-infected cell cultures.

It has numerous advantages, such as ready-to-use DNA and RNA for any downstream analysis, a 40-minute fast purification process, and no toxic reagents. The kit includes columns that can effectively remove DNA and RNA impurities.

Genomic DNA Purification Kit From Blood VIRA02: Virasens Genomic DNA Purification Kit From Blood is designed to purify genomic DNA from fresh or frozen human blood to prepare samples for downstream analysis.

The kit has ready-to-use DNA for any downstream analysis, a fast purification process of 25 minutes, a high-efficiency rate, and no toxic reagents.

Viral RNA Purification Kit VIRA03:  Virasens Viral RNA Purification Kit is designed for researchers to rapidly prepare and purify Viral RNA from cell-free samples such as body fluids, serum, plasma, and supernatant of virally infected cell cultures, especially nasopharyngeal/oropharyngeal swab samples.

This method utilizes membrane spin columns that facilitate rapid and efficient viral particle fragmentation and selectively bind nucleic acids in high concentrations of chaotropic salts. It has high-quality and rapid purification of total viral RNA from respiratory samples and swabs using spin columns. The process requires a minimal amount (150 µl) of sample and provides access to fast, effective, and easy results in a short time.

Genomic DNA Purification Kit From Bacteria VIRA04: Virasens Genomic DNA Purification Kit from Bacteria is designed for in vitro nucleic acid extraction from various gram-positive, gram-negative bacterial species and microorganisms in the blood.

After being extracted from bacterial samples, genomic DNA can be applied to molecular tasks such as PCR, ligation, endonuclease digestion, transformation, cloning, and sequencing. The steps for phenol/chloroform extraction and alcohol precipitation are not included.

Genomic DNA Purification Kit From Tissue VIRA05: The Virasens Genomic DNA Purification Kit From Tissue is specifically designed to isolate nucleic acids from different tissue samples quickly. It utilizes high salt to enhance the binding efficiency of DNA and employs a spin column as the separation matrix for purification.

The key features of this kit are its fast-processing time, efficiency, and ease of use due to the spin-column format. Notably, the kit does not contain any toxic reagents, making it safe to handle. Additionally, it offers scalability, allowing for easy adaptation to varying sample sizes.

Viral DNA Purification Kit VIRA06: The Virasens Viral DNA Purification Kit utilizes a silica-based membrane spin column technology to isolate viral DNA efficiently. The kit offers several advantages, including the absence of toxic reagents like phenol or chloroform. It provides a fast, efficient, and user-friendly process, yielding high amounts of viral DNA for enhanced sensitivity. The purified DNA is suitable for direct use in various procedures, such as RT-PCR, cloning, RFLP analysis, sequencing, qPCR, viral load determination, viral genotyping, and virus detection. Additionally, the kit is compatible with real-time PCR and eliminates the need for precipitation steps, simplifying the workflow.

Plasmid DNA Purification Kit VIRA07: The Virasens Plasmid DNA Purification Kit is specifically designed to isolate high-quality plasmid DNA from cultured bacterial cells rapidly.

The kit offers several advantages, including a fast, efficient, and cost-effective method for plasmid DNA extraction. The resulting plasmid DNA is highly pure, and the extraction process takes only 30 minutes. Moreover, the kit improves plasmid DNA extraction yields, ensuring a reliable and consistent supply of high-quality DNA for downstream applications.

Gel Nucleic Acid Purification Kit VIRA08:  The Virasens Gel Nucleic Acid Purification Kit is made to purify nucleic acid (DNA/RNA fragments) from polyacrylamide/agarose gels with a high yield of recovery. The downstream applications of PCR, restriction digestion, cloning, and transformation can employ pure nucleic acid. The package is free of dangerous compounds like phenol and chloroform.

How to Use DNA and RNA Purification Kits?

The purification process typically involves several key steps, each serving a specific purpose:

1. Cell Lysis: The first step is to disrupt the collected samples’ cell membrane and release the nucleic acids from the cells. This is achieved by using lysis buffers that contain detergents and chaotropic agents. Detergents break down the lipid bilayers of cell membranes, while chaotropic agents disrupt the hydrogen bonding and hydrophobic interactions, helping to solubilize and denature proteins [2].
2. Binding: After cell lysis, the released nucleic acids are in a complex mixture with other cellular components, such as proteins, cellular debris, and lipids. The binding step involves transferring the lysate to a purification column or tube containing a binding matrix, typically silica or magnetic beads. Silica or beads have an affinity for nucleic acids, allowing them to selectively bind to the matrix while other contaminants are removed [2].
3. Washing: Washing steps are performed to remove impurities and contaminants co-purified with the nucleic acids during the binding step. Washing buffers wash away residual proteins, salts, enzymes, and other contaminants, ensuring the purification of nucleic acids [2].
4. Elution: The elution step involves the release of the purified DNA or RNA from the purification matrix. Elution buffers or sterile water are used to disrupt the binding interactions between the nucleic acids and the matrix, allowing the nucleic acids to be collected in a separate tube [2].
5. Quantification and Quality Assessment: This step involves measuring the concentration of the purified DNA or RNA using a spectrophotometer or other quantification methods as specified in the kit’s instructions and assessing the quality of the purified nucleic acids by examining their purity ratios [2].

Overall, DNA and RNA purification kits simplify the process of isolating nucleic acids and yield highly purified DNA or RNA, which are crucial for various molecular biology techniques and genetic analyses.

It is essential to carefully follow the instructions in the DNA and RNA purification kit, as protocols and reagents may vary between kits. The kit’s instructions will provide details on volumes, incubation times, centrifugation speeds, and other critical parameters specific to the kit being used.

Always work in a dedicated and clean laboratory environment, follow proper biosafety guidelines, and wear appropriate personal protective equipment to ensure the integrity of your samples and prevent contamination during the purification process.

REFERENCES

[1] Stadler, J., Lemmens, R., & Nyhammar, T. (). Plasmid DNA purification. The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications, 6(S1), S54-S66.

[2] Shen, J., & Hartmann, E. M. (). DNA extraction protocol for low-biomass environmental samples: adapted from the Lucigen MasterPure Complete DNA and RNA Purification Kit manual.