PCR From Setup to Cleanup

A beginners guide with useful tips and tricks

Sample & Assay Technologies

PCR
The invention of the polymerase chain reaction (PCR) by K. Mullis and co-workers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation, and cancer research, are challenging todays PCR technologies with more demanding requirements. These include the need for increased throughput, higher assay sensitivity, and reliable data analysis. Assay development and evaluation, reproducibility of data, and time to result are still major problems encountered by researchers.

Guidelines for PCR

PCR amplification is performed routinely and thousands of PCR protocols have been developed, yet researchers still encounter technical difficulties with PCR experiments and often fail to obtain specific amplification products. Although there are several different challenges (e.g., smearing, low yield, and nonspecific amplification), there are two main reasons for PCR failure or poor results: the specificity of the reaction and template secondary structure. PCR is both a thermodynamic and an enzymatic process. Successful PCR requires amplification and detection under optimal conditions and each reaction component can affect the result. The annealing step is critical for high PCR specificity. When primers anneal to the template with high specificity, this leads to high yields of specific PCR products and increases the sensitivity of the amplification reaction. However, due to the high primer concentration in the reaction, primers will also hybridize to non-complementary sequences with mismatches. If the primers anneal to the template sequence with low specificity, amplification of nonspecific PCR products and primerdimers may occur. Factors critical in successful PCR include primer design and the reaction chemistry used.

PCR primer design

Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR. The PCR section of the QIAGEN Protocols and Applications Guide provides more information about primer design. The following points should be considered when designing PCR primers and are common to all types of PCR:

Melting temperature (Tm) calculation: 2C x (A+T) + 4C x (G+C) Avoid complementarity in the 23 bases at the 3' end of the primer pairs Avoid mismatches between the 3' end of the primer and the template Avoid runs of 3 or more Cs or Gs at the 3' end of the primer Avoid complementarity within primers and between the primer pair

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 Avoid a T as ultimate base at the 3' end Ensure primer sequence is unique for the template sequence Use a concentration of 0.11.0 M of each primer. For many applications,a primer
concentration of 0.2 M will be sufficient Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/l to avoid repeated thawing and freezing. Store all primer solutions at 20C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.

PCR conditions
The primer and Mg2+ concentration in the PCR buffer and annealing temperature of the reaction may need to be optimized for each primer pair for efficient PCR. In addition, PCR efficiency can be improved by additives that promote DNA polymerase stability and processivity or increase hybridization stringency, and by using strategies that reduce nonspecific primertemplate interactions. Use of high-purity reagents is also essential for successful PCR, especially for amplification of rare templates, for example, single copy genes in genomic DNA or pathogenic viral DNA sequences in genomic DNA isolated from an infected organism. Inclusion of control reactions is essential for monitoring the success of PCR reactions. Wherever possible, a positive control should be included to check that the PCR conditions used can successfully amplify the target sequence. As PCR is extremely sensitive, requiring only a few copies of target template, a negative control containing no template DNA should always be included to ensure that the solutions used for PCR have not become contaminated with the template DNA.
Tip: PCR setup should be performed in a separate area from PCR analysis to ensure that reagents used for PCR do not become contaminated with PCR products. Similarly, pipets used for analysis of PCR products should never be used for setting up PCR.

Primer annealing specificity and PCR buffers

In PCR, annealing occurs between the primers and complementary DNA sequences in the template. Primer annealing must be specific for successful amplification. Due to the high concentration of primers necessary for efficient hybridization during short annealing times, primers can anneal to non-complementary sequences. Amplification of products from nonspecific annealing competes with specific amplification and may drastically reduce the yield of the specific product. The success of PCR largely depends on maintaining a high ratio of specific to nonspecific annealing of the primer molecules. Annealing is primarily influenced by the components of the PCR buffer (in particular the cations) and annealing temperature. Special cation combinations can maintain high primer annealing specificity over a broad range of annealing temperatures. This eliminates the need for optimization of annealing temperatures for each individual primertemplate system and also allows the use of non-ideal PCR assays with different primer annealing temperatures.

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Sample & Assay Technologies

A balanced combination of cations promotes specific primer annealing

Cations in commonly used PCR buffers bind to the negatively charged phosphate groups on the DNA backbone and thereby neutralize these negative charges. This weakens the electrorepulsive forces between the DNA template and primer molecule leading to more stable hybridization of the primer. Most commercially available PCR buffers contain only one monovalent cation, K+, which stabilizes both specific and nonspecific primer annealing. This often results in smearing and nonspecific DNA
Figure 1. Animation on QIAGENs unique PCR buffer system. Watch the video on www.qiagen.com/PCR-video.

amplification, which leads to lower product yields. QIAGEN has found that the balanced combination of K+ and NH4+ used in all QIAGEN PCR buffers provided with all QIAGEN PCR enzyme and master mix kits strongly increases primer annealing specificity. The improved specificity is caused by ammonium ions destabilizing the weak hydrogen bonds at

Stabilization
K+

Destabilization NH 4+
NH3 + H+

Stabilization
K+

mismatched bases (Figures 1 and 2). Discover more about optimized PCR conditions with QIAGENs PCR Kits.

P K

K+

Annealing temperature
The optimal primer annealing temperature is dependent on the base composition (i.e., the proportion of A, T, G, and C nucleotides), primer concentration, and ionic reaction environment. Therefore annealing temperature needs to be optimized for each primer pair. Tip: There is no need to calculate annealing temperature when using QIAGEN PCR kits, as they work over a wide temperature range!

Weak hydrogen band (e.g., between C and T nucleotides) Strong hydrogen band (e.g., between A and T nucleotides)

Figure 2. NH4+ and K+ cations in QIAGEN PCR buffers increases specific primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to non-specific primer template binding over a wide temperature range.

Magnesium ion concentration

Magnesium ions are a critical DNA polymerase cofactor necessary for enzyme activity. Mg2+ binds to DNA, primers, and nucleotides contained in the amplification reaction. The Mg2+ concentration is generally higher than that of dNTPs and primers, and some optimization may be necessary for different template and primer concentrations. A higher than optimal concentration of Mg2+ can stabilize nonspecific binding and is often indicated by decreased yields of specific PCR products and the appearance of background smear or other PCR artifacts.

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PCR additives
Various PCR additives or enhancers are available for improving PCR results. It is claimed that these reagents relieve secondary DNA structure (e.g., hairpin loops in GC-rich regions or in long amplification products), lower template melting temperature, enhance enzyme processivity, stabilize DNA polymerases, or prevent attachment of polymerases to plasticware. Commonly used PCR additives include dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), and glycerol.

Q-Solution
This innovative PCR additive facilitates amplification of difficult templates by modifying the melting behavior of DNA. Use of this unique reagent will often enable or improve suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primertemplate system and is not toxic (Figure 3).
Tip: Q Solution QIAGENs PCR enhancer for difficult templates is found in: QIAGEN Multiplex PCR kits, Type-it kits, QIAGEN Fast Cycling PCR kits, QIAGEN LongRange PCR Kit, HotStar HiFidelity PCR Kits, and with all standard DNA polymerases.

Loading dyes
QIAGENs PCR kits are supplied with CoralLoad PCR Buffer, which has all of the advantages of QIAGEN PCR Buffer, but can also be used to directly load the PCR reaction onto an agarose gel without the need for an additional gel loading buffer. CoralLoad PCR Buffer provides the same high PCR specificity and minimal reaction optimization as the conventional QIAGEN PCR Buffer. Additionally, it contains two marker dyes an orange dye and a red dye that facilitate estimation of DNA migration distance and optimization of agarose gel run time (see Figure 5 on page 13). The buffer ensures improved pipetting visibility and enables direct loading of PCR products onto a gel, for enhanced convenience. Since the PCR results are colored for convenience, for some sensitive downstream applications, it may be necessary to clean up the DNA (see pages 2028 for more information).
Figure 3. Influence of Q-Solution on PCR success. A 4.8 kb fragment was amplified in standard reactions using TopTaq DNA Polymerase with or without Q-Solution. M: marker. Specific amplification was achieved only in reactions containing Q-Solution.
4.8 kb M with Q-Solution without Q-Solution

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Sample & Assay Technologies

Guidelines for degenerate primer design and use

PCR primer sequences are often deduced from amino acid sequences if the exact nucleotide sequence of their target is unknown. However, because of the degeneracy of the genetic code, the deduced sequences may vary at one or more positions. A common solution in these cases is to use a degenerate primer, which is a mixture of similar primers that have different bases at the variable positions. Using degenerate primers can lead to difficulties optimizing PCR assays: within a degenerate primer mixture only a limited number of primer molecules are complementary to the template; the melting temperature (Tm) of primer sequences may vary significantly; and the sequences of some primers can be complementary to those of others. For these reasons, amplification conditions are required that minimize nonspecific primertemplate and primerprimer interactions. The following guidance may help when designing and using degenerate primers. Primer sequence:

Avoid degeneracy in the 3 nucleotides at the 3' end, i.e., if possible

use Met- or Trp-encoding triplets at the 3' end

To increase primertemplate binding efficiency, reduce degeneracy

by allowing some mismatches between the primer and template, especially towards the 5' end, but not the 3' end

Try to design primers with less than 4-fold degeneracy at any given
position Primer concentration:

Begin PCR with a primer concentration of 0.2 M In case of poor PCR efficiency, increase primer concentrations in
increments of 0.25 M until satisfactory results are obtained

Enzymes used in PCR

Several types of thermostable DNA polymerases are available for use in PCR, providing a choice of enzymatic properties. Taq DNA polymerase, isolated from the eubacterium Thermus aquaticus, is the most commonly used enzyme for standard end-point PCR. The robustness of this enzyme allows its use in many different PCR assays. However, as this enzyme is active at room temperature, it is necessary to perform reaction setup on ice to avoid nonspecific amplification. A number of modifications of the original PCR polymerase Taq DNA polymerase are now available for different downstream application
QIAGEN solution: QIAGEN DNA Taq Polymerase for standard and specialized PCR applications

needs, such as hot-start, single-cell, high-fidelity, or multiplex PCR. With an average error rate of 1 in 10,000 nucleotides, Taq DNA polymerase and its variants are less accurate than thermostable enzymes of DNA polymerase family B.

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However, due to its versatility, Taq DNA polymerase is still the enzyme of choice for most routine applications and when used with a stringent hot-start, is suitable for several challenging PCR applications.

Hot-start DNA polymerase

When amplification reaction setup is performed at room temperature, primers can bind nonspecifically to each other, forming primerdimers. During amplification cycles, primerdimers can be extended to produce nonspecific products, which reduces specific product yield. For more challenging PCR applications, the use of hot-start PCR is crucial for successful specific results. To produce hot-start DNA polymerases, Taq DNA polymerase activity can be inhibited at lower temperatures with antibodies or, more effectively, with chemical modifiers that form covalent bonds with amino acids in the polymerase. The chemical modification leads to complete inactivation of the polymerase until the covalent bonds are broken during the initial heat activation step. In contrast, in antibody-mediated hot-start procedures, antibodies bind to the polymerase by relatively weak non-covalent forces, which leaves some polymerase molecules in their active state. This sometimes leads to nonspecific primer extension products that can be further amplified during PCR. These products appear as smearing or incorrectly sized fragments when run on an agarose gel.
QIAGEN solution: HotStarTaq Plus DNA Polymerase for fast and highly specific amplification in all applications

High-fidelity DNA polymerase

Unlike standard DNA polymerases (such as Taq DNA polymerase), highfidelity PCR enzymes generally provide a 3' to 5' exonuclease activity for removing incorrectly incorporated bases. High-fidelity PCR enzymes are ideally suited to applications requiring a low error rate, such as cloning, sequencing, and site-directed mutagenesis. However, if the enzyme is not provided in a hot-start version, the 3' to 5' exonuclease activity can degrade primers during PCR setup and the early stages of PCR. Nonspecific priming caused by shortened primers can result in smearing on a gel or amplification failure especially when using low amounts of template. It should be noted that the proofreading function often causes high-fidelity enzymes to work more slowly than other DNA polymerases. In addition, the A-addition function required for direct UA- or TA-cloning is strongly reduced, resulting in the need for blunt-end cloning with lower ligation and transformation efficiency.
QIAGEN solution: HotStar HiFidelity Polymerase Kit the only high-fidelity kit producing sticky ends for simple UA/ TA cloning procedures for highly sensitive and reliable high-fidelity hot-start PCR

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Sample & Assay Technologies

Optimizing 3' to 5' exonuclease activity

Tip: Proofreading DNA polymerases contain an inherent 3' to 5' exonuclease activity that removes base-pair mismatches. Adding a small amount of a proofreading DNA polymerase to the PCR mixture therefore significantly improves the amplification efficiency of longer PCR products.

Taq DNA Polymerase introduces more errors into the PCR product while copying the template than do so-called proofreading DNA polymerases. Once a mismatch occurs during synthesis, Taq DNA polymerase will either extend the mismatched strand or fall off the template strand, leading to mutated or incomplete PCR products, respectively. Although this does not generally affect PCR efficiency when amplifying shorter PCR fragments, amplification of longer PCR products can be significantly impaired by mismatches introduced during DNA synthesis.

PCR cycling
In theory, each PCR cycle doubles the amount of amplicon in the reaction. Therefore, 10 cycles multiply the amplicon by a factor of ~1000 and so on. Each PCR cycle consists of template denaturation, primer annealing, and primer extension. If the temperatures for annealing and extension are similar, these two processes can be combined. Each stage of the cycle must be optimized in terms of time and temperature for each template and primer pair combination. After the required number of cycles has been completed (see the PCR section of the QIAGEN Protocols and Applications Guide for more information about cycle numbers), the amplified product may be analyzed or used in downstream applications.

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Amplification of long PCR products

Amplification of PCR products longer than 34 kb is often compromised by nonspecific primer annealing, suboptimal cycling conditions, and secondary structures in the DNA template. Lengthy optimization is often necessary, by varying factors such as cycling conditions, primer and dNTP concentrations, and special additives.

Optimizing cycling conditions

While depurination is usually not a problem in standard PCR, it can significantly influence the amplification of longer PCR fragments. This is because longer templates are proportionally more depurinated than shorter ones. For this reason, very short denaturation steps of only 10 seconds give higher yields and no background smearing compared to denaturation steps of 30 seconds or 1 minute (which leads to PCR failure; Figure 4). Extensive depurination is also observed during the final extension step. Therefore, using a lower extension temperature of 68C instead of 72C dramatically improves yield of longer amplification products. The PCR section of the QIAGEN Protocols and Applications Guide provides more information about cycling conditions for longer PCR products.
7.3 kb

QIAGEN solution: QIAGEN LongRange PCR Kit for sensitive and accurate long-range PCR up to 20 kb

A
M 1 2 3

B
M 72C 68C

Figure 4. Effect of cycling conditions. A To illustrate the effects of prolonged heating of the DNA template or the DNA polymerase, three different PCR cycling programs were used to amplify a 7.3 kb fragment of the human interleukin 9 receptor gene. 1: Each PCR cycle had a 60-second denaturation step at 94C. 2: A reaction mixture containing Taq DNA Polymerase but lacking the template DNA was incubated at 94C for 30 minutes; the template DNA was then added and the PCR cycling program started, using 10-second 94C denaturation steps. 3: The PCR cycling program was comparable to 2, except that there was no additional prior incubation. B The 7.3 kb PCR product could only be amplified when the temperature of the extension step was lowered from 72C to 68C. M: markers. Results from duplicate PCR amplifications are shown.

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Sample & Assay Technologies

Frequently asked questions

How can I tell if I have primerdimers in my PCR reaction? In non-quantitative endpoint PCR, primerdimers will appear as a more or less faint smear on an agarose gel, below the product band of interest.

Must I use CoralLoad Gel loading dye when using QIAGENs HotStarTaq Plus DNA Polymerase? No. HotStarTaq Plus DNA Polymerase is supplied with conventional QIAGEN PCR Buffer and CoralLoad PCR Buffer in separate vials. Both buffers minimize nonspecific amplification products, primerdimers, and background. CoralLoad PCR Buffer has all of the advantages of QIAGEN PCR Buffer, but can also be used to directly load the PCR reaction onto an agarose gel without the need to add a gel loading buffer.

How comparable is CoralLoad gel loading dye contained in various QIAGEN PCR Kits to Sigma Red? CoralLoad gel tracking dye contained in Taq, HotStarTaq Plus, TopTaq DNA Polymerase and TopTaq Master Mix Kits separates into 2 fragment-size dependent colors (orange and red) when loaded onto an agarose gel. Sigma Red buffer only has one color which is harder to visualize.

PCR troubleshooting
Comments and suggestions Little or no product or product is multi-banded Pipetting error or missing reagent Repeat the PCR. Check the concentrations and storage conditions of reagents, including primers and dNTP mix. Using the same cycling conditions, repeat the PCR using Q-Solution. Repeat the PCR with different primer concentrations from 0.10.5 M of each primer (in 0.1 M increments). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.

Suboptimal PCR cycling conditions Primer concentration not optimal or primers degraded

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PCR troubleshooting
Comments and suggestions Problems with starting template Check the concentration, storage conditions, and quality of the starting template. If necessary, make new serial dilutions of template nucleic acid from stock solutions and repeat the PCR. Perform PCR with different final concentrations of Mg2+ from 1.55.0 mM (in 0.5 mM increments) using a 25 mM MgCl2 solution. Use 2.5 units of Taq DNA Polymerase per 100 l reaction. Decrease annealing temperature by 2C increments. Annealing time should be between 30 and 60 s. Difficulties in determining the optimal annealing temperature can often be overcome by performing touchdown PCR. Denaturation should be at 94C for 3060 s. or time Ensure that a prolonged initial denaturation is performed as described in the protocols. Try using the hot-start procedure supplied with QIAGEN Taq DNA Polymerase, or for greater specificity, try using HotStarTaq DNA Polymerase. Review primer design When performing PCR in a thermal cycler using a thermal cycler with a heated lid that is switched on, do not overlay the PCR samples with mineral oil, as this may decrease the yield of PCR product.

Mg2+ concentration not optimal

Enzyme concentration too low Incorrect annealing temperature or time

Incorrect denaturation temperature or time

Hot start may be necessary

Primer design not optimal PCR overlaid with mineral oil when using a thermal cycler with a heated lid

Product is smeared Hot start may be necessary Try using the hot-start procedure supplied with QIAGEN Taq DNA Polymerase, or for greater specificity, try using HotStarTaq DNA Polymerase.

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Sample & Assay Technologies

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PCR troubleshooting
Comments and suggestions Too much starting template Check the concentration and storage conditions of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using serial dilutions. If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis. Use 2.5 units of Taq DNA Polymerase per 100 l reaction. Reduce the number of cycles in increments of 3 cycles. See above section for these problems. Follow the specific protocols provided for amplification of long PCR products.

Carry-over contamination

Enzyme concentration too high Too many cycles Mg2+ or primer concentration/design not optimal or primers degraded PCR of long fragments from genomic DNA

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Agarose gel analysis

Agarose gel analysis enables quick and easy quantification of DNA, especially for small DNA fragments (such as PCR products). As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining. The DNA sample is run on an agarose gel alongside known amounts of DNA of the same or a similar size. The amount of sample DNA loaded can be estimated by comparison of the band intensity with the standards either visually or using a scanner or imaging system. Be sure to use standards of roughly the same size as the fragment of interest to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity. CoralLoad PCR Buffer, included in all QIAGEN PCR kits, can be used to directly load the PCR reaction onto an agarose gel separate addition of a gel loading buffer is not required. CoralLoad PCR Buffer provides the same high PCR specificity and minimal reaction optimization as the conventional QIAGEN PCR Buffer. Additionally, it contains two marker dyes an orange dye and a red dye that facilitate estimation of DNA migration distance and optimization of agarose gel run time (Figure 5). The buffer ensures improved pipetting visibility and enables direct loading of PCR products onto a gel, for enhanced convenience. More precise agarose gel quantification can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. In most experiments the effective range for comparative densitometric quantification is between 20 and 100 ng.
Tip: The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve. Figure 5. CoralLoad PCR Buffer. A The buffer contains geltracking dyes for easier pipetting, B enabling immediate gel loading of PCR samples and easy visualization of DNA migration.
A B

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Sample & Assay Technologies

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Running an agarose gel

Agarose gel electrophoresis allows analysis of DNA fragments between 0.1 and 25 kb (e.g., genomic DNA digested with a frequently cutting restriction endonuclease), while pulsefield gel electrophoresis enables analysis of DNA fragments up to 10,000 kb (e.g., undigested genomic DNA or genomic DNA digested with rare cutting restriction endonucleases). The amount of genomic DNA loaded onto a gel depends on the application, but in general, loading of too much DNA should be avoided as this will result in smearing of the DNA bands on the gel.

Gel loading buffer must be added to the samples before loading and serves three main purposes: Tip: If CoralLoad is used, gel loading buffer is not required, as the CoralLoad performs this job!

To increase the density of the samples to ensure that they sink into the wells on loading To add color to the samples through use of dyes such as bromophenol blue or xylene cyanol,
facilitating loading

To allow tracking of the electrophoresis due to co-migration of the dyes with DNA fragments
of a specific size Molecular-weight markers should always be included on a gel to enable analysis of DNA fragment sizes in the samples.

Preparation of samples
1. Add 1 volume of gel loading buffer to 6 volumes DNA sample and mix. Samples should always be mixed with gel loading buffer prior to loading on a gel.

Tip: Do not use sample volumes close to the capacity of the wells, as samples may spill over into adjacent wells during loading. Tip: Be sure that all samples have the same buffer composition. High salt concentrations, for example in some restriction buffers, will retard the migration of the DNA fragments. Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells on loading.

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Agarose gel electrophoresis

1. Apply samples in gel loading buffer to the wells of the gel. Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer. Tip: Make sure that the entire gel is submerged in the electrophoresis buffer. Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip. Tip: Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells. Tip: Be sure to always include at least one lane of appropriate molecular-weight markers.

2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode). Tip: Electrophoresis apparatus should always be covered to protect against electric shocks.

3. Turn on the power supply and run the gel at 170 V with a switch interval of 540 s until the dyes have migrated an appropriate distance. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required. Tip: Avoid use of very high voltages which can cause trailing and smearing of DNA bands in the gel, particularly with highmolecular-weight DNA. Tip: Monitor the temperature of the buffer periodically during the run. If the buffer becomes heated, reduce the voltage. Tip: Melting of an agarose gel during the electrophoresis is a sign that the buffer may have been incorrectly prepared or has become exhausted during the run. Tip: For very long runs, e.g., overnight runs, use a pump to recycle the buffer.

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Sample & Assay Technologies

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Pulse-field gel electrophoresis

1. Apply samples in gel loading buffer to the wells of the gel. Tip: Pulse-field gel electrophoresis uses high voltages, so TBE buffer, which has greater buffering capacity than TAE buffer, should be used. Tip: Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer. Tip: Make sure that the entire gel is submerged in the running buffer. Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip. Tip: Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells. Tip: Be sure to always include at least one lane of appropriate molecular-weight markers. 2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode). Tip: Electrophoresis apparatus should always be covered to protect against electric shocks. 3. Turn on the power supply and run the gel at 170 V with a switch interval of 540 s until the dyes have migrated an appropriate distance. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required. Tip: Monitor the temperature of the buffer periodically during the run. If the buffer becomes overheated, reduce the voltage. Tip: Melting of an agarose gel during the electrophoresis is a sign that the buffer may have been incorrectly prepared or has become exhausted during the run. Tip: For very long runs, e.g., overnight runs, use a pump to recycle the buffer.

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QIAGEN solutions for gel electrophoresis

Gel Pilot Agarose Products for separation of nucleic acid fragments by electrophoresis Gel Pilot DNA Molecular Weight Markers for easy and accurate sizing of DNA fragments Gel Pilot DNA Loading Dye for safe and convenient loading and tracking of DNA samples on electrophoretic gels

Visual analysis of the gel

Staining To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye, e.g., QIAGENs Coral Load PCR Buffer or Gel Pilot DNA Loading Dye. GelPilot DNA Loading Dye is a high-quality gel-loading buffer for analysis of DNA samples using electrophoresis. GelPilot DNA Loading Dye contains 3 different marker dyes (bromophenol blue, xylene cyanol, and orange G) for reliable estimation of DNA migration distance and subsequent optimization of gel run time. One other common dye is the intercalating fluorescent dye ethidium bromide, which can be added either before or after the electrophoresis. Addition of ethidium bromide prior to electrophoresis add ethidium bromide at a concentration of 0.5 g/ml to the melted and subsequently cooled agarose, that is, just before pouring the gel. Mix the agaroseethidium bromide solution well to avoid localized staining. Addition of ethidium bromide after electrophoresis soak the gel in a 0.5 g/ml solution of ethidium bromide (in water or electrophoresis buffer) for 3040 minutes. Tips for handling ethidium bromide:
Note: Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and take appropriate safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not provide full protection. After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals.

Stock solutions of ethidium bromide (generally 10 mg/ml) should be stored at 4C in a dark

bottle or bottle wrapped in aluminum foil

Rinse the gel with buffer or water before examining it to remove excess ethidium bromide Staining buffer can be saved and re-used

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Sample & Assay Technologies

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Avoid the use of ethidium bromide entirely by using CoralLoad PCR Buffer, found in the following QIAGEN kits:

QIAGEN Multiplex PCR Plus Kit QIAGEN Fast Cycling PCR Kit QIAGEN LongRange PCR Kit HotStarTaq Plus DNA Polymerase HotStarTaq Plus Master Mix Kit

Taq DNA Polymerase Taq PCR Core Kit TopTaq DNA Polymerase TopTaq Master Mix Kit

Visualization
Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source. Tip: UV light damages DNA. If DNA fragments are to be extracted from the gel, use a lower intensity UV source if possible and minimize exposure of the DNA to the UV light.

Ethidium bromideDNA complexes display increased fluorescence compared to the dye in solution. This means that illumination of a stained gel under UV light (254366 nm) allows bands of DNA to be visualized against a background of unbound dye. The gel image can be recorded by taking a Polaroid photograph or using a gel documentation system.

Frequently asked questions

Why does my DNA sample float out of the slot when loading it onto an agarose gel? DNA fragments purified with the QIAGEN DNA cleanup systems, (e.g., the QIAquick PCR Purification Kit, the MinElute Reaction Cleanup Kit, the QIAEX II Gel Extraction Kit, etc.) may float out of the loading wells of agarose gels due to residual ethanol carried over from the wash step with Buffer PE (despite the addition of glycerol-containing loading buffer). Use any of the following options to remove residual ethanol from the eluate:

Re-purify the sample using a QIAquick or MinElute column or QIAEX II resin Incubate the eluate at 56C for 10 min to evaporate the ethanol Dry down the sample in a vacuum centrifuge, and resuspend the pellet in a small volume of
sterile water

How should gels be cast with the GelPilot Agarose so that optimal resolution is achieved? When using the GelPilot Agarose, gels should be cast 34 mm thick for optimal resolution of DNA fragments. We also recommend the use of a thin comb (1 mm) to obtain sharper DNA bands.

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How much DNA should be loaded per well of an agarose gel? The amount of DNA per well is variable. The least amount of DNA that can be detected with ethidium bromide is 10 ng. DNA amounts of up to 100 ng per well will result in a sharp, clean band on an ethidium-bromidestained gel. We recommend using GelPilot Agarose for separation of amplified products using agarose gel electrophoresis.

What voltage should be used to run an agarose gel with the GelPilot Agarose? We recommend running agarose gels at 410 volts/cm under horizontal electrophoretic conditions. Higher voltage may result in band streaking, while lower voltage may result in reduced mobility of small (<1000 bp) DNA and diffusion. We recommend using GelPilot Agarose products for separation of amplified products by agarose gel electrophoresis.

What buffer conditions give the best resolution for agarose gel electrophoresis? We recommend the use of 1x TBE buffer for small DNA fragments (<1000 bp) when DNA recovery is not necessary. Gels made using TBE buffer give sharper bands than gels made using TAE buffer. For large DNA fragments of >15000 bp, we recommend the use of 1x TAE buffer. However, since TAE buffer has a lower buffering capacity, it may be necessary to change the buffer when performing electrophoresis for an extended period of time.

Why do I have wavy DNA bands on my agarose gel? Wavy DNA bands on an agarose gel can be caused by dried agarose on the comb teeth. Check the comb teeth for residual dried agarose prior to casting the gel and clean if necessary.

Can I store agarose gel slices containing DNA for gel extraction at a later point? Yes. Cut out the slice of agarose containing the DNA fragment of interest, and store it at 4C in an Eppendorf tube sealed with Parafilm.

How do I separate PCR fragments that are small and very close in size on an agarose gel? The concentration of the agarose gel for separation of multiplex PCR products should be appropriate for the overall size of products generated and can be adjusted for resolving small size differences between PCR fragments. For optimal results, we recommend the use of 1x TAE buffer for preparation and running of the gel. Use the general guidelines listed in the table for choosing the percentage of agarose.

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Minimum difference in size of PCR products >200 bp >100200 bp >50100 bp 2050 bp <20bp*

Maximum size of fragments 2000 bp 1000 bp 750 bp 500 bp 250 bp

Concentration of agarose 1.3% 1.41.6% 1.72.0% 2.53.0% 3.04.0%

* Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard molecular-biology grade agarose. For separation of fragments that differ in size by less than 20 bp, we recommend using high-resolution agarose, for example MetaPhor agarose (FMC Bioproducts). For more information, visit www.cambrex.com. Please see the QIAGEN Multiplex PCR Handbook for additional information and for details on successful multiplex PCR using the QIAGEN Multiplex PCR Kit.

Does QIAGEN have protocols for multiple extractions of DNA fragments from agarose gels? Yes. Follow the Supplementary Protocol High-throughput gel extractions using the QIAquick 96 PCR Purification Kit (QQ03). Contact QIAGEN Technical Services for this protocol.

My sample does not give a fluorescent signal. How do I know whether this is because the PCR did not work or because the target is not expressed? Use a control sample in which the gene of interest is definitely expressed. PCR products that span the region to be amplified in the real-time experiment can also be used as a positive control. Check by agarose gel electrophoresis that the amplification reaction was successful. The quality of the starting template and the integrity of the reagents can be determined by amplifying a housekeeping gene, such as GAPDH or HPRT.

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Cleanup
DNA cleanup
One of the most common tasks in molecular biology is cleaning up nucleic acids from varied matrices to remove buffer salts, enzymes, or other substances that may affect downstream applications, such as cloning, sequencing, microarray analysis, or amplification. There are 3 main areas of DNA cleanup:

Cleanup from enzymatic reactions, e.g., PCR Nucleotide removal Gel extraction and cleanup
A number of cleanup kits are available for different starting samples and downstream application needs. QIAquick and MinElute kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples (Figure 6, next page). Silica-membrane technology eliminates the problems and inconvenience associated with loose resins and slurries. Specialized binding buffers are optimized for specific applications and promote selective adsorption of DNA molecules within particular size ranges.

Cleanup solutions from QIAGEN:

QIAquick PCR Purification Kit for purification of up to 10 g PCR products >100 bp. DNA
of up to 10 kb is purified using a simple and fast bind-wash-elute procedure and an elution volume of 3050 l.

QIAquick Gel Extraction Kit for purification of DNA fragments from gels (up to 400 mg
slices) or enzymatic reactions. DNA ranging from 70 bp to 10 kb is purified using a simple and fast bind-wash-elute procedure and an elution volume of 3050 l.

QIAquick Nucleotide Removal Kit for up to 10 g oligonucleotide (1740mers) and DNA

(40 bp to 10 kb) cleanup from enzymatic reactions. The process uses just three easy steps!

MinElute PCR Purification Kit for purification of up to 5 g PCR products (70 bp to 4 kb) in
low elution volumes using a very simple procedure.

MinElute Gel Extraction Kit for purification of DNA fragments of 70 bp 4 kb from up to

400 mg gel slices. The spin columns are designed to allow elution in very small volumes (as little as 10 l), delivering high yields of highly concentrated DNA.

MinElute Reaction Cleanup Kit for cleanup of up to 5 g DNA (70 bp to 4 kb) from
enzymatic reactions. The kit enables very low elution volumes and uses a fast procedure with easy handling.

QIAEX II Gel Extraction Kit for purification of DNA fragments (40 bp to 50 kb) from gels
and solutions. QIAEX II Suspension is added to solutions or solubilized agarose gel slices and binds DNA in the presence of chaotropic salts before washing and elution.

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2000 bp 500 bp 100 bp 20 mer

Figure 6. Analysis of PCR products before (b) and after (a) purification using the QIAquick PCR Purification Kit. Samples were analyzed on a 1% TAE agarose gel. M: markers.

The advantages of silica-membrane technology include:

QIAquick and MinElute Procedure

PCR or other enzymatic reaction or solubilized gel slice

Yielding high-purity nucleic acids for use in most downstream

applications

Fast and inexpensive No silica-slurry carry over, no alcohol precipitation

QIAcube

QIAGENs QIAquick and MinElute cleanup system use a simple bindwash-elute procedure (Figure 7). Sample mixtures are applied to the spin column (in some applications, this includes a buffer that contains a pH indicator; allowing easy determination of the optimal pH for DNA binding see Figure 8). Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in all subsequent applications.

Vacuum

Vacuum

Optimal pH

pH too High

Pure DNA fragment

From solutions From gels

5min 15min

Figure 7. QIAquick and MinElute procedure. In addition to spin columns, manual and automated high-throughput kits are also available.

Figure 8. pH Indicator Dye. Indicator enables easy checking of the optimal pH. Indicator dye in solubilization and binding buffers (Buffer QG and Buffer PB) identifies optimal pH for DNA binding.

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DNA yield and concentration

DNA yield depends on the following 3 factors:

Elution buffer volume How the buffer is applied to the column Incubation time of the buffer on the column
To completely cover the QIAquick/MinElute membrane, use 100200 l elution buffer. This ensures maximum yield, even when not applied directly to the center of the membrane. Elution with 50 l requires the buffer to be added directly to the center of the membrane, and if elution is done with the minimum recommended volume of 30 l, an additional 1 minute incubation is required for optimal yield. DNA will be up to 1.7 times more concentrated if the column is incubated for 1 minute with 30 l of elution buffer, than if it is eluted in 50 l without incubation. The kits vary depending on the type of reaction and DNA fragment size (e.g., PCR products, gel extraction, enzymatic reactions, nucleotide removal, dye terminator removal) and the required elution volume. Table 1. A guide to enzymatic reaction cleanup using the QIAquick DNA cleanup system
QIAquick PCR Purification Kit QIAquick Nucleotide Removal Kit QIAquick Gel Extraction Kit Tip: Depending on the application, fragment size, etc. it is advisable to use a specialized kit for optimal results. Please consult Tables 1 and 2 to find the kit that best meets your needs.

For DNA cleanup from the following reactions: Alkaline phosphatase cDNA synthesis DNase, nuclease digestion Kinase: DNA fragments Oligonucleotides Ligation Nick translation PCR Random priming Restriction digestion Tailing: DNA fragments Oligonucleotides Specifications Recovery: Oligonucleotides dsDNA Removal: <10mers 1740mers YES YES YES no YES no 100 bp 10 kb 1740mers 40 bp 10 kb 70 bp 10 kb YES YES YES YES no YES YES YES YES YES YES no YES no YES YES YES YES YES no YES YES YES YES YES no YES YES no YES YES no YES YES YES no

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Table 2. Cleanup overview

DNA cleanup application Elution volume <100 bp 100 bp4 kb DNA fragment size 4 kb10 kb 10 kb50 kb

PCR cleanup

3050 l 10 l 20 l

MinElute PCR Purification Kit MinElute 96 UF PCR Purification Kit QIAquick Gel Extraction Kit QIAEX II Gel Extraction Kit MinElute Gel Extraction Kit MinElute Reaction Cleanup Kit QIAquick Nucleotide Removal Kit

QIAquick PCR Purification Kits MinElute PCR Purification Kit MinElute 96 UF PCR Purification Kit QIAquick Gel Extraction Kit QIAEX II Gel Extraction Kit MinElute Gel Extraction Kit QIAquick DNA Cleanup System MinElute Reaction Cleanup Kit QIAquick Nucleotide Removal Kit

QIAquick PCR Purification Kits QIAquick Gel Extraction Kit QIAEX II Gel Extraction Kit QIAquick DNA Cleanup System QIAquick Nucleotide Removal Kit

QIAEX II Gel Extraction Kit QIAEX II Gel Extraction Kit QIAEX II Gel Extraction Kit n.a.

Gel extraction

3050 l >20 l 10 l

Cleanup from enzymatic reactions

3050 l 10 l 30200 l

Nucleotide removal

n.a.: Not applicable.

Frequently asked questions

How can I ensure that I have the right pH? The binding buffer contains a pH indicator, allowing easy determination of the optimal pH for DNA binding (see Figure 8). Binding buffer PB and binding and solubilization buffer QG are specially optimized for use with the QIAquick silica membrane. Buffer QG contains an integrated pH Indicator, while an optional pH Indicator can be added to Buffer PB allowing easy determination of the optimal pH for DNA binding. DNA adsorption requires a pH 7.5, and the pH Indicator in the buffers will appear yellow in this range. If the pH is >7.5, which can occur if during agarose gel electrophoresis, the electrophoresis buffer had been used repeatedly or incorrectly prepared, or if the buffer used in an enzymatic reaction is strongly basic and has a high buffering capacity, the binding mixture turns orange or violet. This means that the pH of the sample exceeds the buffering capacity of Buffer PB or QG and DNA adsorption will be inefficient. In these cases, the pH of the binding mixture can easily be corrected by addition of a small volume of 3 M sodium acetate, pH 5.0, before proceeding with the protocol.

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What is the small band below my fragment of interest on an agarose gel after DNA cleanup using QIAquick? Occasionally, DNA fragments eluted from the silica matrix of QIAquick, MinElute, or QIAEX II Kits will contain denatured single-stranded DNA (ssDNA), appearing as a smaller band on an analytical gel. Under certain conditions, chaotropic agents (present in all silica-based DNA purification methods) can denature DNA fragments. This is a rare event that may be influenced by sequence characteristics such as the presence of inverted repeats or AT-rich stretches. Because salt and buffering agents promote renaturation of DNA strands, the following tips are recommended:

Use the eluted DNA to prepare your downstream enzymatic reaction, but omit the enzyme.
Incubate the reaction mix at 95C for 2 minutes to reanneal the ssDNA, and allow the tube to cool slowly to room temperature before adding the enzyme and proceeding

Alternatively, the DNA can be eluted from the silica-gel membrane or resin in 10 mM Tris buffer
containing 10 mM NaCl. However, the salt concentration of the eluate must then be taken into consideration in downstream applications.

Can I use the QIAquick PCR Purification Kit for restriction enzyme cleanup? Yes. The QIAquick PCR Purification Kit has been used to clean up fragments between 100 bp and 10 kb from a wide range of enzymatic reactions, removing salts, buffers, enzymes, nucleotides, and primers smaller than 40 nucleotides. Reactions that can be cleaned up with the QIAquick PCR Purification Kit include restriction digests, random priming, ligase, kinase, phosphatase, nuclease, nick translation, and cDNA synthesis reactions.

Do you have information about the cleanup of single-stranded DNA (ssDNA) with QIAquick columns? As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions used in the QIAquick and MinElute Kits. Even though no systematic experimental data exists, we expect thatrecovery of ssDNA fragments of approximately 200 nucleotides and belowwill not be very efficient after cleanup using the QIAquick PCR Purification Kit or MinElute PCR Purification Kit. By comparison, it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit. Note that recovery of single strand DNA is influenced to some degreealso by factors such asbase composition and secondary structure. It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results.

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Are Buffer PB of the QIAquick PCR Purification Kit and Buffer QG of the QIAquick Gel Extraction Kit interchangeable? Buffer PB of the QIAquick PCR Purification Kit cannot be used to extract DNA from agarose gels. However, Buffer QG of the QIAquick Gel Extraction Kit can be used to remove salt and proteins from enzymatic reactions by adding 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction and proceeding with step 6 of the Gel Extraction Spin Protocol in the QIAquick Spin Handbook. See the QIAquick Spin Handbookfor a list of reactions which can be cleaned up with the various QIAquick kits.

I bound an 11 kb DNA fragment to a QIAquick column; is it completely lost? Larger DNA fragments bind more tightly to the QIAquick columns. It is difficult to predict whether a DNA fragment larger than 10 kb can be efficiently recovered, because this depends on base composition as well as fragment size. If the fragment is only a few kb larger than the 10 kb limit, it can be helpful to heat the elution buffer EB to 60C and let it incubate on the column for a few minutes before centrifuging. However, please note that it will become less likely to recover your sample the larger the fragment size is. As we cannot guarantee recovery of fragments larger than the maximum cutoff size, we do not recommend purifying such fragments using QIAquick Kits. The QIAEX II Kit can be used to extract DNA fragments up to 50 kb from agarose or polyacrylamide gels.

Cleanup troubleshooting
Comments and suggestions Low or no recovery Buffer PE did not contain ethanol Ethanol must be added to Buffer PE (concentrate) before use. Repeat procedure with correctly prepared Buffer PE. DNA will only be eluted efficiently in the presence of low-salt buffer (e.g., Buffer EB: 10 mM TrisCl, pH 8.5) or water. Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. This is particularly important when using small elution volumes (30 l).

Inappropriate elution buffer

Elution buffer incorrectly dispensed

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Cleanup troubleshooting
Comments and suggestions QIAquick Gel Extraction Kit only Gel slice incompletely solubilized After addition of Buffer QG to the gel slice, mix by vortexing the tube every 23 min during the 50C incubation. DNA will remain in any undissolved agarose. The electrophoresis buffer has been repeatedly used or incorrectly prepared, resulting in a sample pH that exceeds the buffering capacity of Buffer QG and leads to inefficient DNA binding. Add 10 l of 3 M sodium acetate, pH 5.0, to the sample and mix. The color of the mixture will turn yellow indicating the correct pH for DNA binding. Even for binding mixtures with only small color changes (slight orange color), add the 10 l sodium acetate. 7080% recovery can only be obtained from 400 mg gel (>400 mg) slice per QIAquick column. For gel slices >400 mg, use multiple QIAquick columns.

pH of electrophoresis buffer too high (binding mixture turns orange or violet)

Gel slice was too large

QIAquick PCR Purification Kit only Insufficient/no product PCR Estimate DNA recovery by running 10% of PCR product before and after purification on an agarose gel.

QIAquick Gel Extraction Kit and QIAquick PCR Purification Kit only Cloudy and gelatinous appearance of sample mixture after addition of isopropanol This may be due to salt precipitation, and will disappear upon mixing the sample. Alternatively, the gel slice may not be completely solubilized. In this case, apply the mixture to the QIAquick column, centrifuge, and then add 0.5 ml Buffer QG to the column. Let stand for 1 min at room temperature (1525C), and then centrifuge and continue with the procedure. This additional wash will solubilize remaining agarose. The pH in the sample exceeds the buffer capacity of Buffer QG or PB respectively. Add 20 l of 3 M sodium acetate, pH 5.0, to the sample and mix. The color of the mixture will turn yellow indicating the correct pH for DNA binding. Even for samples with slight color changes (orange color), add 10 l sodium acetate.

Binding mixture turns orange or violet

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Cleanup troubleshooting
Comments and suggestions DNA does not perform well (e.g., in downstream ligation reactions) Salt concentration in eluate too high Modify the wash step by incubating the column for 5 min at room temperature after adding 750 l of Buffer PE, then centrifuge. Ensure that the wash flow-through is drained from the collection tube and that the QIAquick column is then centrifuged at 17,900 x g (13,000 rpm) for an additional 1 min.

Eluate contains residual ethanol

QIAquick Gel Extraction Kit only Eluate contaminated with agarose The gel slice is incompletely solubilized or weighs >400 mg. Repeat procedure, including the optional Buffer QG columnwash step.

QIAquick PCR Purification Kit only Eluate contains primerdimers Primerdimers formed are >20 bp and are not completely removed.After the binding step, wash the QIAquick column with 750 l of a 35% guanidine hydrochloride aqueous solution (35 g in 100 ml). Continue with the Buffer PE wash step and the elution step as in the protocol. Use the eluted DNA to prepare the subsequent enzymatic reaction but omit the enzyme. To reanneal the ssDNA, incubate the reaction mixture at 95C for 2 min, and allow the tube to cool slowly to room temperature. Add the enzyme and proceed as usual. Alternatively, the DNA can be eluted in 10 mM Tris buffer containing 10 mM NaCl. The salt and buffering agent promote the renaturation of DNA strands. However, the salt concentration of the eluate must then be considered for subsequent applications.

Eluate contains denatured ssDNA, which appears as a smaller smeared band on an analytical gel

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Discover our complete range of PCR products at www.qiagen.com.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www. qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

Trademarks: QIAGEN, QIAEX, QIAquick, BioRobot, CoralLoad, GelPilot, HotStarTaq, Q-Solution, TopTaq, Type-it (QIAGEN Group); Eppendorf (Eppendorf AG); Parafilm (Bemis Company, Inc.); MetaPhor (FMC Bioproducts). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

2013 QIAGEN, all rights reserved.

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