( ...)HotStarTaq PCR Handbook

For
HotStarTaq DNA Polymerase
HotStarTaq Master Mix Kit

Contents
Kit Contents
Shipping Conditions
Storage and Stability
Technical Assistance
Safety Information
Introduction HotStarTaq DNA Polymerase
PCR Buffer
Q-Solution Product Specifications
Quality Control
PCR Protocols Using: HotStarTaq DNA Polymerase
HotStarTaq DNA Polymerase and Q-Solution
HotStarTaq Master Mix Troubleshooting Strategy
Appendix Starting template
Primer design, concentration, and storage
Number of cycles
Sensitive PCR assays
RT-PCR
Touchdown PCR
Purification of PCR products
Control of contamination Product Warranty and Satisfaction Guarantee
Product Use Limitations
Kit Contents



* Contains 15 mM MgCl2
Contains HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 M each dNTP
Shipping Conditions

HotStarTaq DNA Polymerase is shipped on dry ice but retains full activity at room temperature (1525C) for 2 weeks.
HotStarTaq Master Mix Kit is shipped on dry ice but retains full activity at room temperature (1525C) for 3 days.

Storage and Stability

HotStarTaq DNA Polymerase and HotStarTaq Master Mix Kit, including buffers and reagents, should be stored immediately upon receipt at 20C in a constant temperature freezer. When stored under these conditions and handled correctly, these products can be kept at least until the expiration date (see the inside of the kit lid) without showing any reduction in performance. HotStarTaq Master Mix Kit can also be stored at 28C for up to 2 months.

Technical Assistance

At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products. If you have any questions or experience any difficulties regarding HotStarTaq DNA Polymerase, HotStarTaq Master Mix, or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques.

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDS for each QIAGEN kit and kit component.

24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240

Introduction

HotStarTaq DNA Polymerase has been developed by QIAGEN to provide hot-start PCR for higher PCR specificity. The combination of HotStarTaq DNA Polymerase and the unique QIAGEN PCR Buffer minimizes nonspecific amplification products, primerdimers, and background. It is ideal for amplification reactions involving complex genomic or cDNA templates, very low-copy targets, or multiple primer pairs. HotStarTaq DNA Polymerase makes hot-start PCR simple and easy, eliminating the extra handling steps and contamination risks associated with conventional hot-start methods.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primerdimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15-minute, 95C incubation step, which can easily be incorporated into existing thermal cycling programs. HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product. PCR setup is quick and convenient as all reaction components can be combined at room temperature.

QIAGEN PCR Buffer

The innovative QIAGEN PCR Buffer facilitates the amplification of specific PCR products. During the annealing step of every PCR cycle, the buffer allows a high ratio of specific-tononspecific primer binding. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg²+ concentrations than conventional PCR buffers.* Optimization of PCR by varying the annealing temperature or the Mg²+ concentration is dramatically reduced and often not required.

Q-Solution

The HotStarTaq DNA Polymerase Kit is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent will often enable or improve a suboptimal PCR caused by templates that have a high degree of secondary structure or that are GC-rich. Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, it is nontoxic, and PCR purity is guaranteed. For further information, please read the PCR Protocol Using HotStarTaq DNA Polymerase and Q-Solution.

Product Specifications

Enzyme:
HotStarTaq DNA Polymerase is a modified form of a recombinant 94-kDa DNA polymerase, originally isolated from Thermus aquaticus, cloned in E. coli. (Deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7). One unit of HotStarTaq DNA Polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs into acid-insoluble material within 30 min at 72C, under the assay conditions described in the section Quality Control on the following page.

Concentration: 5 units/l Substrate analogs: dNTP, ddNTP, dUTP, biotin-11-dUTP, DIG-11-dUTP, fluorescent dNTP/ddNTP dNTP Mix: 24 kb/min at 72C Extension rate: 5x concentrated Half-life: Ultrapure quality, PCR-grade 5'3' exonuclease activity: Yes Extra A addition: Yes 3'5' exonuclease activity: No Nuclease contamination: No Protease contamination: No RNase contamination: No Self-priming activity: No Storage and dilution buffer: 20 mM TrisCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% (v/v) Nonidet P-40, 0.5% (v/v) Tween 20, 50% glycerol (v/v), stabilizer; pH 9.0 (20C) Buffers and reagents: PCR Buffer: 10x concentrated. Contains TrisCl, KCl, (NH4)2SO4, 15 mM MgCl2; pH 8.7 (20C). Q-Solution: 5x concentrated MgCl2 solution: 25 mM HotStarTaq Master Mix: 2x concentrated. Contains HotStarTaq DNA Polymerase, PCR Buffer (with 3 mM MgCl2), and 400 M each dNTP.

Quality Control

Enzyme: (See quality-control label inside kit lid for lot-specific values.) Unit assay: Sonicated herring-sperm DNA (12.5 g) is incubated with 0.010.1 units of HotStarTaq DNA Polymerase in assay buffer (25 mM TAPS [tris-(hydroxymethyl)-methyl-aminopropane- sulfonic acid, sodium salt], pH 9.3 at 20C; 50 mM KCl; 2 mM MgCl2; 1 mM DTT; 200 M of each dNTP; 100 Ci [-32P] dCTP) at 72C for 30 minutes. The amount of incorporated dNTPs is determined by precipitation with trichloroacetic acid. HotStarTaq DNA Polymerase is activated by heating for 3 hours at 80C prior to activity measurement. Amplification efficiency assay: The amplification efficiency is tested in parallel amplification reactions and is indicated under Amp. PCR reproducibility assay: PCR reproducibility and specificity are tested in parallel amplification reactions. The reactions must yield a single
specific product. Exonuclease activity assay: Linearized plasmid DNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer. Exonuclease activity per unit of enzyme is indicated under Exo. Endonuclease activity assay: Plasmid DNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer. Endonuclease activity per unit
of enzyme is indicated under Endo. RNase activity assay: RNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer. RNase activity per unit of enzyme is indicated under RNase. RNase activity assay: RNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer. RNase activity per unit of enzyme is indicated under RNase. Protease activity assay: HotStarTaq DNA Polymerase is incubated in storage buffer. Protease activity per unit of enzyme is indicated
under Protease. Self-priming activity assay: Assays are performed under standard PCR conditions, without primers, using HotStarTaq DNA Polymerase and human genomic DNA (purified with the QIAamp DNA Blood Mini Kit). The absence of PCR product is indicated by No under Self-priming. Buffers and Reagents: PCR Buffer, 10x: Conductivity, pH, sterility, and performance in PCR are tested. Q-Solution, 5x: Conductivity, pH, sterility, and performance in PCR are tested. MgCl2, 25 mM: Conductivity, pH, sterility, and performance in PCR are tested. Distilled water: Conductivity, pH, sterility, and performance in PCR are tested. Endonuclease, exonuclease, and RNase activities are tested. HotStarTaq Master Mix Kit: PCR reproducibility assay: The PCR reproducibility assay described above is performed in parallel using HotStarTaq Master Mix and using the separate reagents with the same lot numbers.

PCR Protocol Using HotStarTaq DNA Polymerase

This protocol serves only as a guideline for PCR amplification. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be individually determined.

Notes:

HotStarTaq DNA Polymerase requires an activation step of 15 min at 95C (see step 6 of this protocol).
Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
Use disposable tips containing hydrophobic filters to minimize cross-contamination.
If required, prepare a dNTP mix containing 10 mM of each dNTP. Store this mix in aliquots at 20C.

1. Thaw 10x PCR Buffer, dNTP mix, primer solutions, and 25 mM MgCl2 (if required). It is important to mix the solutions completely before use to avoid localized concentrations of salts.

2. Prepare a master mix according to Table 1. The master mix typically contains all the components needed for PCR except the template DNA. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included. The optimal Mg²+ concentration should be determined empirically but in most cases a concentration of 1.5 mM, as provided in the 1x PCR Buffer, will produce satisfactory results.

Table 1. Reaction composition using HotstarTaq DNA Polymerase



* Contains 15 mM MgCl2

Table 2. Final Mg²+ concentrations



3. Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes. Mix gently, e.g., by pipetting the master mix up and down a few times. It is not necessary to keep PCR tubes on ice as nonspecific DNA synthesis cannot occur at room temperature due to the inactive state of HotStarTaq DNA Polymerase.

4. Add template DNA (<=1 g/reaction) to the individual tubes containing the master mix. For RT-PCR, add an aliquot from the reverse transcriptase reaction. This should not exceed 10% of the final PCR volume (see appendix).

5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 100 l mineral oil.

6. Program the thermal cycler according to the manufacturer's instructions.Each PCR program must start with an initial heat activation step at 95C for 15 min.A typical PCR cycling program is outlined below. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.



7. Place the PCR tubes in the thermal cycler and start the cycling program.

Note: After amplification, samples can be stored overnight at 28C or at 20C for longer storage.

PCR Protocol Using HotStarTaq DNA Polymerase and Q-Solution

This protocol is designed for using Q-Solution in PCR assays. Q-Solution changes the melting behavior of DNA and can be used for PCR systems that do not work well under standard conditions. When using Q-Solution the first time for a particular primertemplate pair, always perform parallel reactions with and without Q-Solution. This recommendation should also be followed if another PCR additive (such as DMSO) was previously used for a particular primertemplate pair.

When using Q-Solution, the following effects may be observed depending on the individual PCR assay:

Case A: Q-Solution enables amplification of a reaction which previously failed.
Case B: Q-Solution increases PCR specificity in certain primertemplate systems.
Case C: Q-Solution has no effect on PCR performance.
Case D: Q-Solution causes reduced efficiency or failure of a previously successful amplification reaction. In this case, addition of Q-Solution disturbs the previously optimal primertemplate annealing. Therefore, when using Q-Solution for the first time for a particular primertemplate system, always perform reactions in parallel with and without Q-Solution.



Notes:
HotStarTaq DNA Polymerase requires an activation step of 15 min at 95C (see step 6 of this protocol).
When using Q-Solution for the first time in a particular primertemplate system, it is important to perform parallel amplification reactions with and without Q-Solution.
Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
Use disposable tips containing hydrophobic filters to minimize cross-contamination.
If required, prepare a dNTP mix containing 10 mM of each dNTP. Store this mix in aliquots at 20C.

1. Thaw 10x PCR Buffer, dNTP mix, primer solutions, and Q-Solution.
It is important to mix the solutions completely before use to avoid localized concentrations of salts. When using Q-Solution, additional MgCl2 is not usually
required.

2. Prepare a master mix according to Table 3.
The master mix typically contains all the components needed for PCR except the template DNA. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included.

Table 3. Reaction composition using HotStarTaq DNA polymerase and Q-Solution



* Contains 15 mM MgCl2

3. Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes.
Mix gently, e.g., by pipetting the master mix up and down a few times. It is not necessary to keep the PCR tubes on ice as nonspecific DNA synthesis cannot occur at room temperature due to the inactive state of HotStarTaq DNA Polymerase.

4. Add template DNA (>=1 g/reaction) to the individual tubes containing the master mix.
For RT-PCR, add an aliquot from the reverse transcriptase reaction. The volume added should not exceed 10% of the final PCR volume (see appendix)
.
5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 100 l mineral oil.

6. Program the thermal cycler according to the manufacturers instructions.
Each PCR program must start with an initial heat activation step at 95C for 15 min A typical PCR cycling program is outlined on the next page. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.

7. Place the PCR tubes in the thermal cycler and start the cycling program.
Note: After amplification, samples can be stored overnight at 28C or at 20C for longer storage.

PCR Protocol Using HotStarTaq Master Mix

This protocol serves only as a guideline for PCR amplification. Optimal reaction conditions, such as incubation times and temperatures, and amount of template DNA, may vary and need to be determined individually.

Notes:
Each PCR program should be started with an initial activation step of 15 min at 95C to activate HotStarTaq DNA Polymerase (see step 6 of this protocol).
HotStarTaq Master Mix provides a final concentration of 1.5 mM MgCl2 in the final reaction mix, which will produce satisfactory results in most cases.
However, if a higher Mg²+ concentration is required, prepare a stock solution containing 25 mM MgCl2.
Set up reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
Use disposable tips containing hydrophobic filters to minimize crosscontamination.

1. Thaw primer solutions.
Mix well before use.
Optional: prepare a primer mix of an appropriate concentration (see Table 4) using the water provided.
This is recommended if several amplification reactions using the same primer pair are to be performed. The final volume of diluted primer mix should be 25 l per reaction including the template DNA, added at step 4.

2. Mix the HotStarTaq Master Mix by vortexing briefly and dispense 25 l into each PCR tube according to Table 4.
It is important to mix the HotStarTaq Master Mix before use in order to avoid localized concentrations of salt. HotStarTaq Master Mix is provided as a 2x concentrate (i.e., a 25 l volume of the HotStarTaq Master Mix is required for amplification reactions with a final volume of 50 l). For volumes smaller than 50 l, the 1:1 ratio of HotStarTaq Master Mix to diluted primer mix and template should be maintained as defined in Table 4. A negative control (without template DNA) should always be included. It is not necessary to keep PCR tubes on ice as nonspecific DNA synthesis cannot occur at room temperature due to the inactive state of HotStarTaq DNA Polymerase.

3. Distribute the appropriate volume of diluted primer mix into the PCR tubes containing the Master Mix.

4. Add template DNA (1g/reaction) to the individual PCR tubes.
For RT-PCR, add an aliquot from the reverse transcription reaction. The volume added should not exceed 10% of the final PCR volume (see appendix).

Table 4. Reaction composition using HotStarTaq Master Mix


*Contains 1.5 mM MgCl2

5. When using thermal cyclers with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 l mineral oil.

6. Program the thermal cycler according to the manufacturers instructions.
Each PCR program must start with an initial heat activation step at 95C for 15 min A typical PCR cycling program is outlined below. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target and primer pair.



7. Place the PCR tubes in the thermal cycler and start the cycling program.
Note: After amplification, samples can be stored overnight at 28C or at 20C for longer storage.

Troubleshooting Strategy

This troubleshooting strategy is a process that has been specifically designed to provide a convenient and time-saving optimization procedure for PCR using HotStarTaq DNA Polymerase or the HotStarTaq Master Mix Kit. It is recommended that the troubleshooting steps are followed in the order they are listed. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology applications (see inside front cover for contact information).

Comments and suggestions

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Little or no product 1. HotStarTaq DNA Polymerase not activated Check whether PCR was started with an initial incubation step at 95C for 15 min. 2. Pipetting error or missing reagent Repeat the PCR. Check the concentrations and storage conditions of reagents, including primers and dNTP mix. In the case of HotStarTaq Master Mix, ensure that a 1:1 ratio of HotStarTaq Master Mix to primertemplate solution is maintained. 3. PCR cycling conditions are not optimal Using the same cycling conditions, repeat the PCR using Q-Solution. 4. Primer concentration not optimal
or primers degraded Repeat the PCR with different primer concen trations from 0.10.5 M of each primer (in 0.1-M steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel. 5. Problems with starting template Check the concentration, storage conditions, and quality of the starting template (see appendix). If necessary, make new serial dilutions of template nucleic acid from stock solutions. Repeat the PCR using the new dilutions. 6. Mg²+ concentration not optimal Perform PCR with different final concentrationsof Mg²+ from 1.55.0 mM (in 0.5 mM steps) using a 25-mM MgCl2 solution (see Table 2). 7. Enzyme concentration too low When using HotStarTaq DNA Polymerase,use 2.5 units per 100 l reaction. If necessary, increase the amount of HotStarTaq DNA Polymerase (in 0.5-unit steps). When using HotStarTaq Master Mix, use 25 l Master Mix per 50 l reaction. 8. Insufficient number of cycles Increase the number of cycles in steps of 5 cycles (see appendix). 9. Incorrect annealing temperature
or time Decrease annealing temperature in 2C steps. Annealing time should be between 30 and 60 seconds. Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing
touchdown PCR (see appendix). 10. Incorrect denaturation temperature
or time Denaturation should be at 94C for 30 to 60 seconds. Ensure that the initial 15-minute 95C incubation step was performed as described in step 6 of the PCR protocols
. 11. Extension time too short Increase the extension time in increments of 1 minute. For PCR using genomic DNA, follow suggestion number 15, below. 12. Insufficient starting template Perform a second round of PCR using a nested PCR approach (see appendix). 13. Primer design not optimal Review primer design (see appendix). 14. RT reaction error For RT-PCR, take into consideration the efficiency of the reverse transcriptase reaction, which averages 1030%. The added volume of reverse transcriptase reaction should not exceed 10% of the final PCR volume (see appendix). 15. PCR of long fragments
from genomic DNA When amplifying products longer than 4 kb from genomic DNA, increase the concentration of genomic DNA in the reaction (see appendix). Alternatively, use the protocol for amplification of long PCR products using ProofStart DNA Polymerase and QIAGEN Taq DNA Polymerase (see the Taq PCR Handbook, March 2002, or the ProofStart PCR Handbook, February 2002). 16. PCR overlaid with mineral oil when using a thermal cycler with a heated lid When performing PCR in a thermal cycler with a heated lid, do not overlay the PCR samples with mineral oil if the heated lid is switched on as this may decrease the yield of PCR product. 17. Problems with the thermal cycler Check the power to the thermal cycler and that the thermal cycler has been correctly programmed. Product is multi-banded 1. PCR cycling conditions not optimal Using the same cycling conditions, repeat the PCR using Q-Solution. 2. Annealing temperature too low Increase annealing temperature in 2C steps. Annealing time should be between 30 and 60 seconds. Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing touchdown PCR (see appendix). 3. Primer concentration not optimal or
primers degraded Repeat the PCR with different primer concen trations from 0.10.5 M of each primer (in 0.1 M steps). In particular, when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel. 4. Primer design not optimal Review primer design Product is smeared 1. Too much starting template Check the concentration and storage conditions of the starting template (see appendix). Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions. When re-amplifying a PCR product, start the re-amplification round using 1 l of a 1-in- 103104 dilution of the previous PCR. In most cases, a nested PCR approach results in higher specificity and sensitivity for reamplification (see appendix). 2. Carry-over contamination If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet 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. 3. Enzyme concentration too high When using HotStarTaq DNA Polymerase, use 2.5 units per 100 l reaction. When using HotStarTaq Master Mix, use 25 l Master Mix per 50 l reaction. 4. Too many cycles Reduce the number of cycles in steps of 3 cycles. 5. Mg²+ concentration not optimal Perform PCR with different final concentrations of Mg²+ from 1.55.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see Table 2). 6. Primer concentration not optimal or
primers degraded Repeat the PCR with different primer concen trations from 0.10.5 M of each primer (in 0.1 M steps). In particular, when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel. 7. Primer design not optimal Review primer design

Appendix

1. Starting template

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification.*

Quality of starting template

Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step enzyme-catalyzed processes. QIAGEN offers a complete range of nucleic acid preparation systems, ensuring the highest-quality templates for PCR, for example the QIAprep system for rapid plasmid purification, the QIAamp and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids, and the RNeasy system for RNA preparation from a variety of sources. For more information about QIAprep, QIAamp, DNeasy, and RNeasy products, contact one of our Technical Service Departments (see inside front cover).

Quantity of starting template

The annealing efficiency of primer to template is an important factor in PCR. Owing to the thermodynamic nature of the reaction, the primer:template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed in Tables 5 and 6 respectively.

Table 5. Spectrophotometric conversions for nucleic acid templates


Absorbance at 260 nm = 1

Table 6. Molar conversions for nucleic acid templates


* Base pairs in haploid genome
For single-copy genes

2. Primer design, concentration, and storage
Standard PCR primers

Prerequisites for successful PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Some general guidelines are given in Table 7

Table 7. General guidelines for standard PCR primers

Length: 1830 nucleotides G/C content: 4060% T_m: Simplified formula for estimating melting temperature (T_m): T_m = 2C x (A+T) + 4C x (G+C) Whenever possible, design primer pairs with similar Tm values. Optimal annealing temperatures may be above or below the estimated T_m. As a starting point, use an annealing temperature 5C below T_m. Sequence: Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primerdimer formation.
Avoid mismatches between the 3' end of the primer and the target-template sequence.
Avoid runs of 3 or more G or C at the 3' end.
Avoid a 3'-end T. Primers with a T at the 3' end have a greater tolerance of mismatch.
Avoid complementary sequences within a primer sequence and between the primer pair.
Commercially available computer software (e.g., Primer Designer 1.0, Scientific Software, 1990; Oligo, Rychlik and Rhoads, 1989) can be used for primer design. Concentration: Spectrophotometric conversion for primers: 1 A260 unit = 2030 g/ml
Molar conversions:
Primer length pmol/g 20 pmol

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18mer
20mer
25mer
30mer 168
152
121
101 119 ng
132 ng
165 ng
198 ng
Use 0.10.5 M of each primer in PCR. For most applications, a primer concentration of 0.2 M will be sufficient. Storage: 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. Degenerate PCR primers

Occasionally, the exact nucleotide sequence of the target-template DNA will not be known, for instance when it has been deduced from an amino acid sequence. To enable such templates to be amplified by PCR, degenerate primers can be used. These are actually mixtures of several primers whose sequences differ at the positions that correspond to the uncertainties in the template sequence. Hot-start PCR using HotStarTaq DNA Polymerase often improves the specificity of PCR amplifications that employ degenerate primers by reducing the formation of nonspecific PCR products and primerdimers. Table 8 gives recommendations for further optimizing PCR using degenerate primers. Table 9 shows the codon redundancy of each amino acid.

Table 8. Guidelines for design and use of degenerate primers

Sequence: Avoid degeneracy in the 3 nucleotides at the 3' end.
If possible, use Met- or Trp-encoding triplets at the 3' end.
To increase primertemplate binding efficiency, reduce degeneracy by allowing mismatches between the primer and template, especially towards the 5' end (but not at the 3' end).
Try to design primers with less than 4-fold degeneracy at any given position. 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.
Table 9. Codon redundancy

Amino acid Number of codons

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Met, Trp
Cys, Asp, Glu, Phe, His, Lys, Asn, Gln, Tyr
Ile
Ala, Gly, Pro, Thr, Val
Leu, Arg, Ser 1
2
3
4
6
3. Number of cycles

A cycling program usually consists of between 25 and 35 cycles, depending on the number of copies of the starting template. Too many cycles do not necessarily lead to a higher yield of PCR product; instead they may increase nonspecific background and decrease the yield of specific PCR product. Table 10 provides a general guideline for choosing the number of cycles.

Table 10. General guidelines for choosing the number of PCR cycles


*Refer to Table 6 to calculate the number of molecules. When starting with cDNA templates, it is important to take into account the efficiency of reverse transcription in cDNA synthesis, which is on average 1030%.
Refers to single-copy genes.

4. Sensitive PCR assays

PCR can be performed to amplify and detect just a single copy of a nucleic acid sequence. However, amplification of such a low number of target sequences is often limited by the generation of nonspecific PCR products and primerdimers. The combination of HotStarTaq DNA Polymerase and QIAGEN PCR Buffer increases specificity both at the start of and during PCR. Thus HotStarTaq DNA Polymerase is well suited to such highly sensitive PCR assays.

Nested PCR

If PCR sensitivity is too low, a nested PCR method can increase PCR product yield. Nested PCR involves two rounds of amplification reactions. The first-round PCR is performed according to the PCR Protocol using HotStarTaq DNA Polymerase. Subsequently, an aliquot of the first-round PCR product, for example 1 l of a 1-in-103104 dilution, is subjected to a second round of PCR. The second-round PCR is performed with two new primers that hybridize to sequences internal to the first-round primertarget sequences. In this way, only specific first-round PCR products (and not nonspecific products) will be
amplified in the second round. Alternatively, it is possible to use one internal and one firstround primer in the second PCR; this method is referred to as semi-nested PCR.

Single-cell PCR

HotStarTaq DNA Polymerase has been shown to successfully amplify a single-copy gene from just a single cell. The recommendations provided in Table 11 are intended to serve as a starting point for performing such a single-cell PCR from genomic template DNA. If the PCR product is undetectable or the product yield is too low, perform a nested PCR.

Table 11. Recommendations for single-cell PCR

Isolation and storage of single cells: Single cells may be isolated by different methods (e.g., by flow cytometry or by micromanipulation).
Keep samples cool during the cell-isolation procedure to prevent DNA degradation.
Transfer cell into a PCR tube that has been filled with 20 l of 1x PCR Buffer. Immediately freeze the sample on dry ice.
Store cell at 80C until required for PCR analysis. PCR setup: Prepare a fresh master mix for single-cell PCR (see below).
Thaw the cells on ice.
Distribute 30 l of the master mix into each PCR tube, and place the tubes in the thermal cycler. Immediately start the cycling program with a 10 min incubation step at 95C to activate HotStarTaq DNA Polymerase for single-cell PCR. 50 cycles of PCR may be required to amplify a single-copy gene in one round of PCR. Master mix preparation: Prepare a master mix that has a final volume of 30 l per PCR, as detailed below.
Notes:
Addition of carrier nucleic acid is usually required (e.g., E. coli 5S rRNA).
Use polyacrylamide gel- or HPLC-purified primers only. Component Volume/reaction Final concentration

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10x PCR Buffer*
25 mM MgCl2
10 mM dNTP

Primer A
Primer B
5S ribosomal RNA (E. coli)
HotStarTaq DNA Polymerase
Distilled water
Single cell in 1x PCR Buffer 3 l
Variable
1 l

Variable
Variable
Variable
1 l
Variable
20 l 1x

200 M of
each dNTP
0.2 M
0.2 M
50 ng/reaction
5 units/reaction

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Total volume 50 l Multiplex PCR

Multiplex PCR is a demanding technique that requires extensive optimization of the amounts of Taq DNA Polymerase, MgCl2, additional reagents, and primers. Often, cycling parameters need to be changed. In many cases, results are still disappointing and further optimization is required. QIAGEN now offers the QIAGEN Multiplex PCR Kit, which eliminates the need for optimization, making the development of multiplex PCR assays both simple and fast. The QIAGEN Multiplex PCR Kit is the first kit specifically developed for multiplex PCR and provides an easy-to-use master-mix format. QIAGEN Multiplex PCR Master Mix contains pre-optimized concentrations of HotStarTaq DNA Polymerase and MgCl2, plus dNTPs and an innovative PCR buffer specially developed for multiplex PCR. The new PCR buffer contains a balanced combination of salts and additives, which enables comparable efficiencies for annealing and extension of all primers in the reaction. The kit is highly suited for multiplex PCR applications such as typing of genetically modified animals and plants, microsatellite analysis, determination of bacteria and viruses, or amplification of regions carrying SNPs. For more information about the QIAGEN Multiplex PCR Kit, contact your local QIAGEN Technical Services or distributor (see inside front cover).

5. RT-PCR
To perform PCR using RNA as a starting template, the RNA must first be reverse transcribed into cDNA in a reverse transcriptase reaction (RT reaction). Failure of the subsequent PCR is often a result of the limitations of the RT reaction. On average, only 1030% of the original RNA molecules is reverse transcribed into cDNA. The expression level of the target RNA molecules and the relatively low efficiency of the reverse transcription reaction must
be considered when calculating the appropriate amount of starting template for subsequent PCR. The volume of the RT reaction transferred should not exceed 10% of the total PCR volume. General guidelines are presented in Table 12 below.

Table 12. General guidelines for performing RT-PCR

RNA purification
and reverse
transcription: QIAGEN offers the RNeasy system for total RNA isolation, Oligotex Kits for messenger RNA isolation, and Omniscript Reverse Transcriptase for reverse transcription.* Follow the detailed protocol in the Omniscript Reverse Transcriptase Handbook. Or, when using an enzyme from another supplier, follow the manufacturers instructions. The following guidelines may be helpful.

Mix the following reagents in a microcentrifuge tube:
4.0 l 5x RT buffer
1.0 l RNase inhibitor (5 units/l)
2.0 l DTT (0.1 M)
1.0 l each dNTP (10 mM)
~1 g RNA
2.5 l primer (0.2 g/l)
reverse transcriptase
Add RNase-free water to a final volume of 20 l.
Incubate following the manufacturers instructions.
Heat the reaction mix to 95C for 5 min to inactivate the
reverse transcriptase. PCR: Prepare a PCR mixture following steps 13 in protocols.
Add 25 l from the RT reaction to each PCR tube containing the master mix.
Continue with step 5 in the PCR protocols. Oligotex is not available in Japan.
* For further information about RNeasy, Oligotex, and Omniscript products, contact your local QIAGEN Technical Services or distributor (see inside front cover).
Please refer to the manufacturers instructions for the amount of enzyme required.

6. Touchdown PCR

Touchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of PCR. The annealing temperature in the initial cycle should be 510C above the Tm of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 12C/cycle until a temperature is reached that is equal to, or 25C below, the Tm of the primers. Touchdown PCR enhances the specificity of the initial primertemplate duplex formation and hence the specificity of the final PCR product.

To program your thermal cycler for touchdown PCR, you should refer to the manufacturers instructions.

7. Purification of PCR products

After amplification, the PCR sample contains a complex mixture of specific PCR product and residual reaction components such as primers, unincorporated nucleotides, enzyme(s), salts, mineral oil, and probably nonspecific amplification products. Before the specific PCR product can be used in subsequent experiments it is often necessary to remove these contaminants. The QIAquick system offers a quick and easy method for purifying the final PCR product. Using the MinElute system, PCR products can be purified in higher concentrations due to the low elution volumes needed in this system. For more information about QIAquick and MinElute products, please call QIAGEN Technical Services or your local distributor (see inside front cover).

8. Control of contamination

It is extremely important to include at least one negative control that lacks the template nucleic acid in every PCR setup to detect possible contamination.

General physical precautions

Separate the working areas for setting up the PCR master mix and DNA handling, including the addition of starting template, PCR product analysis, or plasmid preparation. Ideally, use separate rooms.

Use a separate set of pipets for the PCR master mix. Use of pipet tips with hydrophobic filters is strongly recommended.

Prepare and freeze small aliquots of primer solutions and dNTP mix. Use of fresh distilled water is strongly recommended.

In case of contamination, laboratory benches, apparatus, and pipets can be decontaminated by cleaning them with a 1/10 dilution of a commercial bleach
solution.* Afterwards, the benches and pipets should be rinsed with distilled water.

General chemical precautions

PCR stock solutions can also be decontaminated using UV light. This method is laborious, however, and its efficiency is difficult to control and cannot be guaranteed. We recommend storing solutions in small aliquots and using fresh aliquots for each PCR.

Contamination by PCR product carry-over can be eliminated by using the commercially available uracil-N-glycosylase (UNG). The procedure involves substituting dUTP for dTTP in the PCR setup and treating all PCR mixtures with UNG prior to PCR amplification. As a result, any PCR product containing dUTP carried over from previous rounds of amplification is destroyed by cleavage during the initial incubation step that activates HotStarTaq DNA Polymerase.

Another approach to preventing amplification of contaminating DNA is to treat individual reaction mixtures with DNase I or restriction enzymes that cut between the binding sites of the amplification primers used, before adding the template DNA sample.

* Most commercial bleach solutions are approximately 5.25% sodium hypochlorate. Sodium hypochlorate is an irritant and should be handled with caution.

Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department. We will credit your account or exchange the product as you wish.
A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see inside front cover).

Product Use Limitations

HotStarTaq DNA Polymerase and HotStarTaq Master Mix Kit are developed, designed, and sold for research purposes only. They are not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use. All due care and attention should be exercised in the handling of many of the materials described in this text.





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Related biology technology :

1. HotStarTaq DNA Polymerase
2. QIAGEN Multiplex PCR Handbook
3. TransMessenger Transfection Reagent Handbook
4. Transfection Reagent Selector Kit Handbook