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DNA gel extraction: chemical contaminants

DNA gel extraction: chemical contaminants


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I am performing a gel extraction to purify DNA after a double digest with EcoRI and BamHI. After the gel extraction I need to complete a ligation step before bacterial transformation. The problem I am having is that the DNA after the gel extraction have some kind of chemical contaminant that won't allow me to quantify the DNA using the Nanodrop spectrophotometer. I am getting very low 260/230 ratios.

Has anyone had some issues with this?

I am using the Qiagen Qiaquick Gel Extraction Kit.


Here are some possible reasons:

  1. Your yield is very low, which you can infer from a low 260 absorbance. Either make sure your restriction digest is efficient or add more substrate to the digest reaction.
  2. Carbohydrate contamination, which will cause high 230 absorbance. Wash the column one more time with buffer QG (the step is described in the Qiagen gel extraction manual; if you're already doing a wash with buffer QG, do an extra one).
  3. You didn't dry the column enough and some ethanol from the wash remains in the sample. This can also cause high 230 absorbance and your sample with smell of ethanol. Place your column in an empty collection tube and spin it down for a long time (15-20 min). Don't worry, the DNA can withstand it.

10 Tips for Better DNA Gel Extraction Results

Anyone who has worked in a molecular biology lab knows that DNA gel extraction can be surprisingly challenging. Why is this? Is it because of poor product yields, or maybe it’s because the gel extraction process uses harsh chemicals and conditions (e.g., chaotropic salts, ethidium bromide, ethanol, heat) that will damage or denature DNA and potentially decrease cloning success.

In this article we share some tips to help you maximize your yields of high quality DNA from the gel extraction process.


Monarch® DNA gel extraction kits and components, New England Biolabs

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DNA isolation is a critical step in molecular biology. It is necessary to obtain a specific DNA fragment from the extracted DNA in molecular biology techniques. After isolating plasmids it may contain some chromosomal DNA contamination it will interrupt the further processing of cloning. So it is better to recover the plasmid DNA by eluting it from agarose gels (extraction). The first step in extracting DNA is identifying the DNA band which is to extract, by illuminating under UV light. The desired band is then carefully cut by a Scalpel blade. There are several methods for extracting DNA from the agarose gels. Recovery of DNA from agarose gels by electrophoresis onto DEAE-cellulose membrane is one of the rapid and effective methods. Electroelution is also a good method for DNA recovery especially for larger DNA fragments. Several kit methods are also used in laboratories. In electro elution, the gel fragment of desired DNA band is placed into a dialysis bag with buffer. The bag is then placed into a gel box containing buffer and subjected to an electric current. The DNA extracted is precipitated from the solution. In another recovery method using DEAE cellulose membrane, the gel piece is slide into the slit of DEAE cellulose paper which will bind the DNA. Then an electric current is applied in order to move the band in the paper. DNA is washed off from the paper and is precipitated with ethanol. Freeze-thaw method of extraction is a commonly used advantageous DNA recovering method which will supports the common laboratory facilities. It is very simple and easy to perform with good yield.


Most of the molecular biology laboratories use Low melting point agarose for the separation of DNA from agarose. Low melting point agarose melts at a lower temperature than standard agarose and this temperature does not denature double stranded DNA. It is better to extract DNA fragments in a TAE buffered gel than TBE buffered gel because borate present in the buffer interferes with purification methods.


Unique Column Design

Not only are Monarch columns designed to use less plastic, but they are also optimized to eliminate buffer retention Monarch columns are designed without a retaining ring, thus preventing salts from carrying over into your eluted samples. The tapered design of the column also enables low volume elution (&ge 6 µl).

Monarch DNA Cleanup Column Design
Monarch columns are designed for performance
Monarch columns are designed without a frit, which eliminates buffer retention and the risk of carryover contamination, providing fast, worry-free DNA purification.

Efficient DNA Purification from Gels and Enzymatic Reactions

Monarch kits enable efficient purification of highly-pure and highly concentrated DNA from enzymatic reactions and agarose gels. Purified DNA is ready for use in downstream manipulations such as ligations and restriction digests.

Purify oligos and small DNA fragments

The Monarch PCR & DNA Cleanup Kit protocol can be modified to enable the purification of ssDNA, oligonucleotides, and other small DNA fragments. The Oligonucleotide Cleanup protocol efficiently removes unincorporated nucleotides, short oligos, dyes, enzymes, and salts from labeling and other enzymatic reactions. The modified protocol utilizes the same columns and bind/wash/elute workflow of the Monarch PCR & DNA Cleanup Kit with > 70% recovery and cleanup of oligonucleotides &ge 15 bp (dsDNA) or &ge18 nt (ssDNA).

Recovery of ssDNA and dsDNA oligonucleotides (1 µg) using the Oligonucleotide Cleanup Protocol and the Monarch PCR & DNA Cleanup Kit.

Videos

Monarch ® DNA Gel Extraction Kit protocol

Learn how to extract DNA from agarose gels using the Monarch DNA Gel Extraction Kit.

Tips for using the Monarch ® DNA Gel Extraction Kit

Optimize your DNA gel extractions with our quick tips for using the Monarch DNA Gel Extraction Kit.

How to recycle your Monarch ® Kit components

Learn how you can easily recycle all of the components in your Monarch Kits.


What are the factors that affect DNA agarose gel electrophoresis?

  1. Nucleic acid sample- Type, purity and quantity.
  2. Buffer- concentration and pH of buffer and buffer type.
  3. Electric field- voltage applied current and charge of particles.
  4. Other- gel preparation, gel concentration, other chemicals.

Type of sample:

Varied techniques of gel electrophoresis are used for protein, DNA and RNA separation. Though the basic technique of separation remains the same, minor changes are needed.

RNAs are smaller than the DNA, thus migrate faster than DNA in a gel. So to run an RNA gel, we need a gel with a different concentration (we are not talking about protein here).

The concentration of a gel for DNA and RNA varies. Sample type also has a significant effect on gel electrophoresis results. The concentration of gel, preparation technique, running time and other factors vary depending upon the sample types.

Purity of nucleic acid:

Mishandling and inappropriate DNA extraction cause serious problems in PCR and electrophoresis. To get good results, DNA should be nearly pure. A DNA with a 260/280 ratio of approximately 1.80 is highly recommended.

To run the gDNA, pure DNA migrates properly in a gel, contrary, the migration pattern of contaminated DNA isn’t interpretable.

Two common contaminants of DNA extraction are RNA and protein. A smear below DNA and a smear above DNA band are observed when get contaminated with protein and RNA, respectively. Pure DNA separates evenly, beautifully and distinguishably in a gel.

If you wish to achieve good yield and quality of DNA this article is for you: How to Increase DNA Purity and Yield?

Contaminated DNA gel is seen in the figure below,

Contaminated and inappropriate gel electrophoresis results.

Concentration of DNA:

Concentration also matters to separate DNA perfectly. The concentration of DNA and the size of gel pores have a relation! Larger DNA fragments can’t migrate efficiently from a smaller sized gel.

Therefore the concentration of DNA is important for separating DNA and getting good results. For example, to run genomic DNA we need 0.8% gel while to run PCR amplicons, we need 2.0% gel.

(can you tell me which percent gel is required for restriction digestion?)

Sometimes, highly concentrated DNA can’t separate on a gel, we need to dilute it, although diluting the sample decreases the concentration but not purity. Ideally, a 0.8% gDNA gel can separate 500 to 900μg DNA.

Type of buffer:

Buffer is an essential ingredient of the electrophoresis, as it provides a constant liquid medium and pH during the run. TAE and TBE are two common systems widely used during gel electrophoresis of the DNA.

However, both don’t have the same activity. A newbie can use either but for the specialized experiment or for critical investigations, the expert needs to choose the best from either.

The Borat can react with the sugar portion of the DNA and influence the migration, consequently and therefore not recommended by experts. Tris-acetate-EDTA is the first choice to run DNA, worldwide.

A pH of the buffer:

pH is as important as other factors, as minor changes in the pH of the buffer adversely influence the migration of DNA and gel results, consequently. pH 8.3 is ideal for running DNA perfectly.

Composition of the buffer:

The chemical composition of TAE buffer is Tris, acetate and EDTA and the chemical composition of TBE buffer is Tris, borate and EDTA. Each component executes a vital role to control separation, so their concentration and quantity matter a lot!

We have covered an article on this topic. You can check out the chemical preparation here: Agarose gel electrophoresis buffer .

Change in composition causes slower migration, DNA degradation or other problems in gel electrophoresis. So you need to weigh and prepare each buffer correctly. Read the above article.

Current and voltage:

The current and voltage of the run affect the migration and separation of biomolecules. When the applied current is too high, it makes DNA run faster, heats the gel and buffer and makes DNA smear.

DNA can’t separate correctly.

When the applied current is too low, DNA can’t run from the gel pores and diffuses in a gel. Fragments of DNA can’t be separated in either case. Ideally, 80 to 100v current is advisable to run a DNA gel. Note that the voltage applied also depends on the size of DNA fragments.

The gDNA is run by 60 to 80V whilst PCR amplicons are run by 100 to 120V, ideally. Take the advice of an expert before deciding the current and voltage of the gel. The current of electrophoresis should also remain constant during the whole run to run DNA at a constant speed.

The concentration of agarose:

Agarose is an ingredient of a DNA gel. It is boiled in the buffer to make it gel. Every DNA sample is different, a varied concentration of agarose is needed to run different samples.

Concentration of Agarose

PCR product and plasmid DNA

If the concentration of agarose and quantity of DNA can’t be maintained fragments can’t be separated efficiently.

Gel preparation:

Superior expertise is though not required to prepare a gel, gel preparation makes a huge difference. If the gel isn’t settled, having air bubbles, not evenly distributed and the thickness isn’t good, our DNA can’t migrate accurately.

Air bubbles stop DNA running, and is one of the most common gel preparation problems beginners do. One needs so much practice to do it perfectly.

Another factor is gel thickness and inappropriate distribution. A gel caster should be balanced first before pouring the gel. In addition to this, the quantity of gel must be sufficient enough too.

Evenly distributed gel having 1 to 1.5cm thickness is recommended. If the thickness is too low, DNA molecules can’t migrate at a constant rate.

Handling errors:

  • Inaccurate chemical preparation.
  • Haphazard weighing and solution preparation
  • Inaccurate pipetting
  • Inaccurate pH balance
  • Handling errors in DNA extraction
  • Inaccurate sample preparation

Your hard work and accuracy reflect in your work, how you prepared and run a gel therefore every step should be performed sincerely. Handling errors aren’t accepted for detection of mutation, validating experiments and disease diagnosis.

Pipetting chemicals and solution are yet another common mistakes that influence results of DNA gel electrophoresis. Learn correct pipetting technique, understand when to use double press and when to use the triple press. Besides, other factors are loading DNA samples in wells, inappropriate labeling of samples, preparing BPB dye, etc.


Recovering DNA from gel - and then secquencing it? (Sep/26/2008 )

i was wondering how to optimize DNA extraction from an agarose gel so that i can secquence a band. i use the qiagen gel extraction kit that says that it gives high yields of purified DNA apt for secquencing, neverthless, my secquences come back awful. any trick, i dunno, optimal agarose concentration, sybr vs ethidium bromide, etc?
i need to solve this problem ASAP
thnx!

We routinely extract bands from agarose gels using the Qiagen kit and send them for sequencing, usually without problems. The only thing we do that is different from the kit is to elute in water, rather than the provided buffer (which I believe is just Tris, pH 8.0).

Are you measuring the amount of template DNA you're using? What do your sequences look like? Are you using the same primers for PCR and for sequencing? How clean is the PCR reaction from which you're isolating your band? What is the Tm of your primers? How long are they?

Personally I don't recommend dna extraction from gel for sequencing or cloning, unless you got 2 or more bands never got really good results and apparently what I see in the forums many have same problem. If you have a single band use a spin column assay for cleaning the product. If not that lucky and have several bands, then use a good low melting agarose, if possible buy band cutter (so you can work faster and get less agarose as possible). Other thing that can try is using a pair of primers that are more inside for the sequence reaction.

hi,
my secquences basically have a very high background, the lady at the sequencer here blames it on my 260/230 ratio. also, they dont resemble what im supposed to be seing at all! im sure the band im cutting is the correct one, as this worked well for the person who was in charge of this project before me (he left for a postdoc abroad and i am now in charge of this project). i have pictures of his gels clearly indicating the band and they are identical to mine.
i suspect maybe the gels here have to much bromide ethidium, i will try this weeked with sybr. i will also try designing nested primers, but this is a tricky viral sequence which will be hard to get that easily! my primers have a very low tm, maybe that could also account for the problem, but as i said before, this worked very well for the reasearcher before me (to whom i worte and doesnt have a clue of what could be going wrong and seems to be to busy to help here)
thanks a lot guys!

Another possibility - TA clone your PCR product, do colony PCR to make sure you have the right thing, then send your clone for sequencing.

yeah, thats what i thought in the first place, but i have to do over a hundred of these during this semestre and my lab dpesnt really have enough cash for all the topo kits, anitbiotics, plasmid extraction kits, etc.
advisor said just send the product to sequencing and forget all those "fancy" intermediate steps.

yeah, thats what i thought in the first place, but i have to do over a hundred of these during this semestre and my lab dpesnt really have enough cash for all the topo kits, anitbiotics, plasmid extraction kits, etc.
advisor said just send the product to sequencing and forget all those "fancy" intermediate steps.

If 260/230 is the problem. clean the reaction with EtoH precipitation. because 230 normally indicate salt contamination. i got this problem often whenever i m doing gel extraction. too much salt is NEVER good for sequencing . dissolve in H20 after that. then u good to go!

Make sure the amount of DNA is correct. Trouble can arise from either too much or too little DNA in the sequencing reaction, and is probably the single largest issue. Modern sequencers are friendlier to small amounts of DNA, but the reaction can fail dramatically with too much. You could try further purification of your DNA, but I would first make sure that you really had the amount you thought you did. You could try reprecipitating the DNA, making sure to do the 70% ethanol washes of the pellet. Salt contamination of the sample can also cause problems. Describe how you are purifying the DNA after cutting it from the gel.


Abstract

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.

Citation: Psifidi A, Dovas CI, Bramis G, Lazou T, Russel CL, Arsenos G, et al. (2015) Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples. PLoS ONE 10(1): e0115960. https://doi.org/10.1371/journal.pone.0115960

Received: September 25, 2014 Accepted: November 28, 2014 Published: January 30, 2015

Copyright: © 2015 Psifidi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: The data is all contained within the paper.

Funding: This research was funded by the Seven Framework Program of the European Commission, project 3SR (Sustainable Solutions for Small ruminants). 3SR Grant agreement no.: FP7-KBBE-2009-3-245140. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Weird Band gel migration - Molecular biology (Nov/22/2005 )

Hello, My problem is that after a PCR, I run in a gel for purifying the fragment of interest. I cut the band of interest and after I extract the DNA with a kit extraccion gel. Is very surprising what I see after extraction. I run the purified single band in a gel and 2 bands appear, one with the correct size and the other one smaller (the smaller one with quite intensity, almost as the correct one). My PCR product is very rich in A+T, the size is 1,6Kb, I think that could be secondary structure in the DNA, loops or thing like that. I think this structure doesn´t form in the PCR buffer reaction (NaCl, Mg) (in the first gel) but after extraccion the DNA is in water and form 2 structures. It is possible? Is any other reason?
Thanks a lot
It make me crazy

Just a thought, but have you checked that your gel purification reagents are free of contaminating DNA? I don't know if DNA contamination would give such an intense band, but it could explain why you cut one band out and get two after purification.

<have you checked that your gel purification reagents are free of contaminating DNA?>

it couldn´t be contamination because I am doing this with 4 different size PCR (but similar sequence homology AT rich) and all 4 after purification presents 2 bands, and the strange one is the different size. The most surprising thing is that the 4 PCR products are between 1,5 Kb and 1,7 Kb, and the distant between the correct band and the strange one is the same in all of them.
Look at the file

I would'nt worry too much I've seen that with pCR products purified by PEG precipitation the migration profile but the sequencing doesn't revealed any mixed inputs it's just a migration problem.

How did you extract your band ?

Did you try a restriction cut to see whether you hacve the correct profile ?

what is your template for your PCR reaction. if it is RNA , it could be that you are seeing isoforms if any for your product of interest, since they are forming a basic pattern in all the four primers. just a thought.

How did you extract your band ?
Kiagen gel extraction kit

Did you try a restriction cut to see whether you hacve the correct profile ?
No, But I´m going to try it

what is your template for your PCR reaction?

The template is genomic DNA

Yes, I think it is just a migration problem and there is not mixed of inputs.

How did you extract your band ?
Kiagen gel extraction kit

Did you try a restriction cut to see whether you hacve the correct profile ?
No, But I´m going to try it

what is your template for your PCR reaction?

The template is genomic DNA

Yes, I think it is just a migration problem and there is not mixed of inputs.

OK just try the restriction digest and will see . I use routinely the Quiagen kit without any trouble but who knows with molecular biology

<< just try the restriction digest and will see>>

I did the restriction digest and it is correct. It is not any strange band. Pesji was right, It was only a migration problem.
Thanks a lot to everyone



Comments:

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  2. Wicasa

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  3. Drue

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  4. Gokinos

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  5. Chilton

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  6. Voshakar

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