Sample to Insight

PCR – From Setup to CleanupA beginner’s guide with useful tips and tricks!

2 PCR – From Setup to Cleanup 10/2015

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 today’s 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

primer–dimers 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:

• The melting temperature (Tm) can be calculated according to the formula:

2°C x (A+T) + 4°C x (G+C)

• Avoid complementarity in the 2–3 bases at the 3’ end of the primer pairs

PCR – From Setup to Cleanup 10/2015 3

• 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

• Avoid a T as ultimate base at the 3’ end

• Ensure primer sequence is unique for the template sequence

• Use a concentration of 0.1–1.0 μM of each primer. For many applications, a primer

concentration of 0.2 μM is 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/μl to

avoid repeated thawing and freezing. Store all primer solutions at –20°C. 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 primer–template

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 known to contain the target sequence 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.

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.

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.

4 PCR – From Setup to Cleanup 10/2015

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 primer–template system

and also allows the use of non-ideal PCR assays with different primer

annealing temperatures.

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

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

mismatched bases (Figures 1 and 2).

Discover more about optimized PCR conditions with QIAGEN’s PCR Kits.

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!

Figure 1. Animation on QIAGEN’s unique PCR buffer system. Watch the video on www.qiagen.com/PCR-video.

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.

NH3 + H+

DestabilizationNH4

+

Stabilization

K+

P– B H

H

H

H

K+

P– B

Stabilization

K+

P–B

K+

P–B

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

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

PCR – From Setup to Cleanup 10/2015 5

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.

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 primer—template system and is not toxic (Figure 3).

Loading dyes

Many QIAGEN’s 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

Tip: Q Solution — QIAGEN’s PCR enhancer for difficult templates — is found in: QIAGEN Multiplex PCR kits, QIAGEN OneStep RT-PCR Kit, Type-it® kits, QIAGEN Fast Cycling PCR kits, QIAGEN LongRange PCR Kit, HotStar HiFidelity PCR Kits, and with all standard DNA polymerases.

6 PCR – From Setup to Cleanup 10/2015

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 7 on page 17). 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 24—34 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.

M with Q-Solution without Q-Solution

– 4.8 kb

PCR – From Setup to Cleanup 10/2015 7

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 that minimize nonspecific primer–template and primer–primer

interactions are required. The following guidance may help when designing and using degenerate primers are required.

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 primer–template 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 μM

• In case of poor PCR efficiency, increase primer concentrations in

increments of 0.25 μM 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. QIAGEN solution: QIAGEN DNA Taq Polymerase — for standard and specialized PCR applications

8 PCR – From Setup to Cleanup 10/2015

A number of modifications of the original “PCR polymerase” — Taq

DNA polymerase — are now available for different downstream

application 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. 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 primer–dimers.

During amplification cycles, primer–dimers 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 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.

High-fidelity DNA polymerase

Unlike standard DNA polymerases (such as Taq DNA polymerase),

high-fidelity 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

QIAGEN solution: HotStarTaq® Plus DNA Polymerase — for fast and highly specific amplification in all applications

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

PCR – From Setup to Cleanup 10/2015 9

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.

Optimizing 3’ to 5’ exonuclease activity

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.

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.

Figure 4. High-resolution analysis of multiplex PCR samples PCR products were generated using the QIAGEN Multiplex PCR Kit according to the standard protocol. PCR samples (13 µl) were analyzed [A] on a 2% agarose gel and [B] and [C] using the QIAxcel system with the QIAxcel DNA High Resolution gel cartridge and the preinstalled OM500 method. [B] The gel image produced by the QIAxcel system shows much higher resolution than the agarose gel. [C] Each sample lane can be visualized individually in electropherogram form. Lane 7 is shown.

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

A B

C

1 2 3 4 5 6 7 8 9 10 NTC 1 2 3 4 5 6 7 8 9 10

10 PCR – From Setup to Cleanup 10/2015

Amplification of long PCR products

Amplification of PCR products longer than 3–4 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. Our LongRange PCR kits

overcome the need to develop specific long-range PCR protocols by

providing a dedicated and highly reliable solution for amplification of

extremely long PCR product (up to 40 kb DNA).

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). Extensive depurination is also observed during the final extension

step. Therefore, using a lower extension temperature of 68°C instead of

72°C 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.

Successful multiplex PCR

Multiplex PCR allows simultaneous amplification of multiple DNA targets

in the same reaction. This increases throughput, reduces reagent costs

and conserves precious sample material. Multiplex PCR offers many

advantages for end‐point PCR applications, including genotyping from

genomic DNA and gene expression analysis from mRNA derived

cDNA. It also permits co‐amplification of an internal positive control

with sequences of interest in the same reaction, allowing sample

preparation and PCR to be monitored and the absence of inhibitors to

be confirmed. The internal control can either be an endogenous gene

QIAGEN Multiplex PCR Kit – for highly specific and sensitive multiplex PCR without optimization requirements.

PCR – From Setup to Cleanup 10/2015 11

(e.g., a housekeeping gene) or an exogenous nucleic acid spiked into

the reaction.

QIAGEN’s multiplex PCR chemistry enables unlimited success with as

many targets as you like and is provided in an easy‐to‐use master mix

format. The master mix contains a novel PCR buffer and pre‐optimized

concentrations of HotStarTaq DNA Polymerase, MgCl2 and dNTPs. The

master mix also contains Factor MP, which further facilitates multiplex

amplification by increasing and stabilizing the local concentration of

primers at the template. These pre‐optimized reagents conveniently

remove the need to optimize reaction conditions or cycling parameters.

Template

Primer

Destabilizationof mismatchedprimer by NH4

+

Template

Primer Stabilizationby Factor MP

Nonspeci�c primer annealing

Speci�c primer annealing

Figure 5. Unique Type‐it Microsatellite PCR Buffer promotes stable and efficient annealing. A. NH4

+ ions prevent nonspecific primers from annealing to the template. Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. B. Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.

12 PCR – From Setup to Cleanup 10/2015

Capillary electrophoresis – an advanced alternative

Capillary electrophoresis offers an excellent, non‐hazardous alternative to traditional gel analysis of DNA and RNA, while

streamlining your workflow and reducing time to results. Nucleic acid molecules are size separated by applying a current

to a gel‐filled capillary and detected as they migrate past a detection spot. The signal data is then transmitted through a

photomultiplier tube and converted into an electropherogram for further analysis.

The QIAxcel® Advanced System — for effortless DNA and RNA analysis

The QIAxcel Advanced System fully automates sensitive, high‐resolution

capillary electrophoresis of up to 96 samples per run (Figure 6). DNA

fragment analysis of 12 samples can be performed in as little as

3 minutes. The QIAxcel Advanced offers a high degree of flexibility

by providing four different ready-to-run gel cartridges that match most

analysis requirements (see Table 1). These cartridges also allow samples

to be analyzed with a minimum of hands-on interaction, reducing

handling errors and eliminating the need for tedious gel preparation.

Only a few, simple steps are required to operate the system: load the

gel cartridge, fill and load the buffer tray, load your samples in 96‐well

plates or in PCR tubes or strips and select the process profile. Within

minutes of starting a run, you can see the first results appearing in real

time on the computer screen. Comprehensive data reports are easily

generated, saved or exported to meet your needs, facilitating integration

of the system in your daily routine.

With a resolution down to 3–5 bp for fragments smaller than 500 bp,

the QIAxcel Advanced System ensures greater accuracy than slab‐gel

methods. Sample consumption is less than 0.1 μl per run, saving

precious sample for downstream analysis. Used in combination with

proven QIAGEN PCR kits, this system provides an all‐in‐one solution for

reproducible, standardized PCR fragment analysis, ensuring significant

time and cost savings.

A B

Gel matrix with dyePositivecharge

Capillary

Nucleic acid with dye

LED light source

DetectorPhoto-

multiplier

QIAxcel ScreenGel Software

Negativecharge

Figure 6. An innovative principle. A. QIAxcel gel cartidge. B. The capillary electrophoresis principle is similar to that used in sequencers. Nucleic acid molecules are size separated by applying a current to a gel‐filled capillary and detected as they migrate toward the positively charged terminus. The signal data pass through a photomultiplier and are converted to an electropherogram and gel image by QIAxcel ScreenGel Software.

* Best resolution between 1 – 3 kb. † Run time depends on method used.

QIAxcel Kit Analyte Size range 100 bp – 500 bp 500 bp – 1 kb 1 kb – 5 kb 5 kb – 10 kb

Run time/

12 samples†

QX DNA High Resolution Kit DNA 15 bp – 10 kb 3 – 5 bp 50 bp 200 – 500 bp 1 – 1.5 kb 7 – 20 min

QX DNA Screening Kit DNA 15 bp – 5 kb 20 – 50 bp 50 – 100 bp 500 bp – 5 min

QX DNA Fast Analysis Kit DNA 15 bp – 3 kb 50 – 100 bp 100 – 250 bp 250 bp – 1 kb* – 3 – 5 min

QX RNA QC Kit v2.0 RNA 15 bp – 6 kb – – – – 10 min

Table 1. QIAxcel Kit speci�cations

Best resolution

PCR – From Setup to Cleanup 10/2015 13

Frequently asked questions

How can I tell if I have primer–dimers in my PCR reaction?

In non-quantitative endpoint PCR, primer–dimers 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 QIAGEN’s 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,

primer–dimers 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.

14 PCR – From Setup to Cleanup 10/2015

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.

Suboptimal PCR cycling conditions Using the same cycling conditions, repeat the

PCR using Q-Solution.

Primer concentration not optimal or primers

degraded

Repeat the PCR with different primer concentrations

from 0.1–0.5 μM of each primer (in 0.1 μM

increments). In particular, when performing highly

sensitive PCR, check for possible degradation of

the primers on a denaturing polyacrylamide gel.

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.

Mg2+ concentration not optimal Perform PCR with different final concentrations of

Mg2+ from 1.5–5.0 mM (in 0.5 mM increments)

using a 25 mM MgCl2 solution.

Enzyme concentration too low Use 2.5 units of Taq DNA Polymerase per

100 μl reaction.

PCR troubleshooting

PCR – From Setup to Cleanup 10/2015 15

Comments and suggestions

Incorrect annealing temperature or time Decrease annealing temperature by 2°C

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.

Incorrect denaturation temperature or time Denaturation should be at 94°C for 30–60 s.

Ensure that a prolonged initial denaturation is

performed as described in the protocols.

Hot start may be necessary Try using the hot-start procedure supplied with

QIAGEN Taq DNA Polymerase, or for greater

specificity, try using HotStarTaq Plus DNA

polymerase.

Primer design not optimal Review primer design

PCR overlaid with mineral oil when using a

thermal cycler with a heated lid

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.

PCR troubleshooting

16 PCR – From Setup to Cleanup 10/2015

Comments and suggestions

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.

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.

Carry-over contamination 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.

Enzyme concentration too high Use 2.5 units of Taq DNA Polymerase per

100 μl reaction.

Too many cycles Reduce the number of cycles in increments of

3 cycles.

Mg2+ or primer concentration/design not

optimal or primers degraded

See above section for these problems.

PCR of long fragments from genomic DNA Follow the specific protocols provided for

amplification of long PCR products.

PCR troubleshooting

PCR – From Setup to Cleanup 10/2015 17

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 most 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 7).

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.

CoralLoad PCR Buffer is 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

Figure 7. CoralLoad PCR Buffer. A. The buffer contains gel-tracking dyes for easier pipetting, B. enabling immediate gel loading of PCR samples and easy visualization of DNA migration.

Tip: The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve.

A B

18 PCR – From Setup to Cleanup 10/2015

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 pulse-field 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:

• 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.

Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells

on loading.

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.

PCR – From Setup to Cleanup 10/2015 19

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.

2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode).

3. Turn on the power supply and run the gel at 170 V with a switch interval of 5–40 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: 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.

Tip: Electrophoresis apparatus should always be covered to protect against electric shocks.

Tip: Avoid use of very high voltages which can cause trailing and smearing of DNA bands

in the gel, particularly with high-molecular-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.

20 PCR – From Setup to Cleanup 10/2015

Pulse-field gel electrophoresis

1. Apply samples in gel loading buffer to the wells of the gel.

2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode).

3. Turn on the power supply and run the gel at 170 V with a switch interval of 5–40 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: 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.

Tip: Electrophoresis apparatus should always be covered to protect against electric shocks.

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.

PCR – From Setup to Cleanup 10/2015 21

Visual analysis of the gel

Staining

To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye,

such as ethidium bromide or SYBR® Green. SYBR Green is considered to be less hazardous, with

respect to mutagenicity, than ethidium bromide.

Addition of ethidium bromide prior to electrophoresis — add ethidium bromide at a concentration

of 0.5 μg/ml to the melted and subsequently cooled agarose, that is, just before pouring the gel.

Mix the agarose–ethidium bromide solution well to avoid localized staining.

Addition of ethidium bromide after electrophoresis — soak the gel in a 0.5 μg/ml solution of

ethidium bromide (in water or electrophoresis buffer) for 30–40 minutes.

Tips for handling ethidium bromide:

• Stock solutions of ethidium bromide (generally 10 mg/ml) should be stored at 4°C 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

Visualization

DNA stained with SYBR Green or ethidium bromide displays increased fluorescence compared

to the dye in solution. This means that illumination of a stained gel under UV light (254–366 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.

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.

22 PCR – From Setup to Cleanup 10/2015

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.

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 56°C 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 so that optimal resolution is achieved?

Gels should be cast 3–4 mm thick for optimal resolution of DNA fragments. We also recommend

the use of a thin comb (1 mm) to obtain sharper DNA bands.

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-bromide–stained gel.

What voltage should be used to run an agarose gel?

We recommend running agarose gels at 4–10 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.

PCR – From Setup to Cleanup 10/2015 23

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 4°C in a

microcentrifuge 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.

* 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.

Maximum size of fragments Concentration of agarose

>200 bp

2000 bp 1.3%>100–200 bp 1000 bp 1.4–1.6%

>50–100 bp 750 bp 1.7–2.0%

20–50 bp 500 bp 2.5–3.0%

<20bp* 250 bp 3.0–4.0%

Minimum difference in sizeof PCR products

24 PCR – From Setup to Cleanup 10/2015

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.

What is the QIAxcel Advanced System?

The revolutionary QIAxcel Advanced System is a fully automated one-step capillary electrophoresis

system that replaces traditional, labor-intensive gel analysis of DNA and RNA.

• Rapid analysis of up to 96 samples without manual intervention

• Safety and convenience with ready-to-use gel cartridges

• Robust results for nucleic acid concentrations as low as 0.1 ng/μl

• Standardized and accurate analysis with a resolution down to 3–5 bp

• User-friendly analysis software that supports 21 CFR Part 11 compliance

For a virtual demonstration of the software, features and applications of the QIAxcel Advanced

System visit: www.qiagen.com/QIAxcelAdvancedDemo

What is the shelf life of the QIAxcel cartridges?

All QIAxcel DNA and RNA Kits can be stored for a guaranteed minimum time of 135 days after

delivery. QIAxcel DNA and RNA ready-to-use cartridges are reusable and can process up to

2400 samples.

What is the minimum sample volume required for the QIAxcel Advanced?

For the QIAxcel System and QIAxcel Advanced, the minimum sample volume required to guarantee

sample injection in each channel is 10 μl. Robust results can be achieved with sample concentration

as low as 0.1 ng/μl of nucleic acid; you can dilute your sample without loss of signal quality.

PCR – From Setup to Cleanup 10/2015 25

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 8, 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 μg 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 30–50 μl.

• 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 30–50 μl.

• QIAquick Nucleotide Removal Kit — for up to 10 μg oligonucleotide (17–40mers) 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 μg 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 μl), delivering high yields of highly concentrated DNA.

• MinElute Reaction Cleanup Kit — for cleanup of up to 5 μg DNA (70 bp to 4 kb) from

enzymatic reactions. The kit enables very low elution volumes and uses a fast procedure with

easy handling.

26 PCR – From Setup to Cleanup 10/2015

• 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.

The advantages of silica-membrane technology include:

• Yielding high-purity nucleic acids for use in most downstream

applications

• Fast and inexpensive

• No silica-slurry carry over, no alcohol precipitation

QIAGEN’s QIAquick and MinElute cleanup system use a simple

bind-wash-elute procedure (Figure 9). 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 10).

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.

–

–

– –

Maaa bbbM

2000 bp

500 bp

20 mer100 bp

Optimal pH pH too High

Figure 8. 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.

Figure 10. 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.

Figure 9. QIAquick and MinElute procedure. In addition to spin columns, manual and automated high-throughput kits are also available.

Vacuum

Vacuum

QIAquick and MinElute Procedure

PCR or otherenzymatic reaction orsolubilized gel slice

Pure DNA fragment

QIAcube

From solutions 5minFrom gels 15min

PCR – From Setup to Cleanup 10/2015 27

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 100–200 μl elution buffer. This ensures

maximum yield, even when not applied directly to the center of the membrane. Elution with

≤50 μl 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 μl, 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 μl of elution buffer, than if it is eluted in 50 μl 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.

Tip: Depending on the application, fragment size, etc. it is advisable to use a specialized kit for optimal results. Please consult Tables 2 and 3 to find the kit that best meets your needs.

QIAquick PCR Puri�cation Kit QIAquick Nucleotide Removal Kit QIAquick Gel Extraction Kit

For DNA cleanup from thefollowing reactions:

YES no noYES

cDNA synthesisAlkaline phosphatase YES YES

YESKinase:DNase, nuclease digestion YES YES

no YES noYES

OligonucleotidesDNA fragments YES YES

YES YES YESYES

Nick translationLigation YES YES

YESPCRRandom priming

no noYES YES YES

YES YES YES

YESTailing:Restriction digestion YES YES

DNA fragments

Recovery:Speci�cations

Oligonucleotides no YES no

100 bp – 10 kb 40 bp – 10 kb 70 bp – 10 kb—

dsDNAOligonucleotides 17–40mers —

Removal: <10mers YES YES YES

YES17–40mers no no

Table 2. A guide to enzymatic reaction cleanup using the QIAquick DNA cleanup system

28 PCR – From Setup to Cleanup 10/2015

DNA cleanup application Elution volume<100 bp 100 bp–4 kb 4 kb–10 kb 10 kb–50 kb

—

DNA fragment size

30–50 µl

— —10 µl

— —20 µl

PCR cleanup

—30–50 µl

>20 µl

— —10 µl

Gel extraction

—30–50 µl

— —10 µl

QIAquick PCRPuri�cation Kits

QIAquick PCRPuri�cation Kits

QIAEX II GelExtraction Kit

MinElute PCRPuri�cation Kit

MinElute PCRPuri�cation Kit

MinElute 96 UFPCR Puri�cation Kit

MinElute 96 UFPCR Puri�cation Kit

QIAquick GelExtraction Kit

QIAquick GelExtraction Kit

QIAquick GelExtraction Kit

QIAEX II GelExtraction Kit

QIAEX II GelExtraction Kit

QIAEX II GelExtraction Kit

QIAEX II GelExtraction Kit

MinElute GelExtraction Kit

MinElute GelExtraction Kit

QIAquick DNACleanup System

QIAquick DNACleanup System

QIAEX II GelExtraction Kit

MinElute ReactionCleanup Kit

MinElute ReactionCleanup Kit

QIAquickNucleotideRemoval Kit

QIAquickNucleotideRemoval Kit

QIAquickNucleotideRemoval Kit

n.a.30–200 µl

Cleanup fromenzymatic reactions

Nucleotide removal

n.a.: Not applicable.

Table 3. Cleanup overview

PCR – From Setup to Cleanup 10/2015 29

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 10). 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.

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 A–T-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 95°C 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

30 PCR – From Setup to Cleanup 10/2015

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

that recovery of ssDNA fragments of approximately 200 nucleotides and below will 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 degree also by factors such as

base 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.

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 Handbook for 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 60°C 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 polyacrylamidegels.

PCR – From Setup to Cleanup 10/2015 31

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.

Inappropriate elution buffer DNA will only be eluted efficiently in the presence

of low-salt buffer (e.g., Buffer EB: 10 mM Tris·Cl,

pH 8.5) or water.

Elution buffer incorrectly dispensed 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 μl).

QIAquick Gel Extraction Kit only

Gel slice incompletely solubilized After addition of Buffer QG to the gel slice, mix

by vortexing the tube every 2–3 min during

the 50°C incubation. DNA will remain in any

undissolved agarose.

pH of electrophoresis buffer too high (binding

mixture turns orange or violet)

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 μl 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

10 μl sodium acetate.

Cleanup troubleshooting

32 PCR – From Setup to Cleanup 10/2015

Cleanup troubleshooting

Comments and suggestions

Gel slice was too large 70–80% recovery can only be obtained from

≤400 mg gel (>400 mg) slice per QIAquick

column. For gel slices >400 mg, use multiple

QIAquick columns.

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 (15–25°C), and then centrifuge and

continue with the procedure. This additional

wash will solubilize remaining agarose.

Binding mixture turns orange or violet The pH in the sample exceeds the buffer capacity

of Buffer QG or PB respectively. Add 20 μl 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 μl sodium acetate.

PCR – From Setup to Cleanup 10/2015 33

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 μl of Buffer PE, then centrifuge.

Eluate contains residual ethanol 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.

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 column-wash step.

QIAquick PCR Purification Kit only

Eluate contains primer–dimers Primer–dimers formed are >20 bp and are not

completely removed. After the binding step,

wash the QIAquick column with 750 μl 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.

Cleanup troubleshooting

34 PCR – From Setup to Cleanup 10/2015

Cleanup troubleshooting

Comments and suggestions

Eluate contains denatured ssDNA, which

appears as a smaller smeared band on an

analytical gel

Use the eluted DNA to prepare the subsequent

enzymatic reaction but omit the enzyme. To

reanneal the ssDNA, incubate the reaction

mixture at 95°C 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.

PCR – From Setup to Cleanup 10/2015 35

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®, Sample to Insight®, QIAxcel®, QIAEX®, QIAquick®, CoralLoad®, HotStarTaq®, Q-Solution®, TopTaq®, Type-it®, (QIAGEN Group); Parafilm® (Bemis Company, Inc.); MetaPhor® (FMC Bioproducts); SYBR® (Life Technologies Corporation). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

PROM-6053-004 © 2015 QIAGEN, all rights reserved.

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