Low Contaminant Amplification of DNA Using MTP™ Taq DNA Polymerase (D7442)

General Laboratory Considerations

Every precaution should be taken to avoid contamination of reagents with unknown/unwanted DNA.
This includes the following:

  • Use a “clean area” for the setup of PCR reactions.2 A clean area is a separate lab space (such as a hood) preferably in a separate lab, both of which are free from PCR products. The clean area should contain dedicated lab coats and pipettes (see below) and should be cleansed (either with 10% bleach or UV lamp) after each use. Amplified PCR products should never enter a clean area.
  • Transfer amplified PCR reactions to another area for analysis.
  • Use sterile, aerosol barrier pipette tips to minimize the risk of aerosol contamination. Change tips after each single use.
  • Change gloves frequently, especially after handling DNA.

Procedure

Amplification Procedure
The optimal conditions for the concentration of MTP Taq DNA Polymerase, template DNA, primers, and MgCl2 will depend on the system being utilized. It may be necessary to determine the optimal conditions for each individual component. It is recommended that the enzyme and the MgCl2 be titrated to determine the optimal efficiency if the below protocol is shown to be less than satisfactory. Sigma offers a separate PCR Optimization Kit, Catalog Number OPT2, that contains a variety of buffers and adjuncts for optimizing the specificity, fidelity, and yield of a PCR product.


1. Add the following reagents to a suitable PCR tube/plate in the following order:

 

Volume Reagent Final Concentration
w µL Water
5 µL 10x MTP Taq buffer 1x
1 µL 10 mM dNTP mix 200 µM each dNTP
x µL Forward Primer (typically 15-30 bases in length) 0.1–0.5 µM
y µL Reverse Primer (typically 15-30 bases in length) 0.1-0.5 µM
0.5 µL MTP Taq DNA Polymerase 0.05 units/µL
z µL Template DNA (typically 10 ng) 200 pg/µL
50 µL* Total reaction volume  

*Maintain final concentrations when scaling reaction volumes. Preparing enzyme / nucleotide / buffer master mixes either with or without primers or template facilitates the setup of multiple reactions.

 

2. Vortex gently to mix. Briefly centrifuge to collect reaction on the bottom of the tube.

3. If using a thermal cycler without a heated lid, add 50 µL of mineral oil to the top of each tube to prevent evaporation.

4. Cycle using thermocycler of choice. The amplification parameters may require optimization for individual primers, templates, and thermal cyclers.

Common cycling parameters are:

Step Temperature Time Cycles
Denature 94 °C 1 minute 1
Denature 94 °C 1 minute 30
Anneal 5 ºC below primer Tm 15 - 60 seconds
Extend 72 °C 30 - 65 seconds/kb
Cool 4 °C ≥ 0 seconds 1

 

5. Amplified DNA can be evaluated by standard methods (e.g. agarose gel electrophoresis).3 If used, mineral oil may be removed by a single 50 µL chloroform extraction. .

Troubleshooting Guide

Problem Possible Causes Solution
No PCR product Missing or degraded PCR component(s) Repeat reaction, including positive controls to ensure all components are functioning. Use a checklist to mark off each reaction component as it is added.
Insufficient PCR cycles Increase the number of PCR cycles (3-5 additional cycles at a time)
Annealing temperature too high Decrease annealing temperature in 2-4 °C increments. Use a temperature gradient thermocycler.
Sub-optimal primer design Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primer to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%. We recommend the use of a primer selection program such as Oligo4 or primer3.
Insufficient template First try increasing cycle number. If that fails, repeat reaction with >5-fold higher concentration of template.
Poor-quality template Evaluate the template integrity by agarose gel electrophoresis. It may be necessary to repurify template using methods that minimize nicking and shearing.3 For amplification of damaged template unable to be amplified with standard enzymes, try Sigma's DNA repair and amplification enzyme blend Restorase® , Catalog Number R1028.
Insufficient enzyme 0.5 µL (2.5 units) is sufficient for most applications. It is recommended that the cycling parameters be optimized before the enzyme concentration is increased. In rare cases, yields can be improved by increasing the enzyme concentration. However, if the enzyme amount is above 1.0 µL (5.0 units), higher background levels may be seen.
Inappropriate denaturation temperature Optimize denaturation temperature by increasing or decreasing the temperature in 1 °C increments or on a temperature gradient thermocycler.
Denaturation time may be too long or too short Optimize the denaturation time by increasing or decreasing it in 10‑second increments
Insufficient extension Increase extension time. Generally, increasing the extension time 1 minute per 1 kb is sufficient; however, longer extension times may be beneficial.
Sub-optimal [Mg++] This is unlikely if the 10X reaction buffer (provided) is used and the deoxynucleotides do not exceed a concentration of 0.2 mM each. However, if the concentration of EDTA in the sample is greater than 5 mM, this can reduce the effective concentration of magnesium.5
Sub-optimal [dNTPs] This is unlikely if the final concentration of each deoxynucleotide is 0.2 mM. This concentration of dNTPs is suitable for a wide range of applications. If the dNTPs are being prepared in the laboratory, be sure that the final concentration of each deoxynucleotide is 0.2 mM. If the concentration of dNTPs is increased, the Mg2+ concentration will need to be increased proportionately.5
Inherently difficult target template In many cases, targets that are difficult to amplify contain unusually high GC content and/or secondary structure.6 2-Betaine has been reported to help amplification of high GC content templates at a concentration of 0.8-1.3 M.7 In some cases, the addition of 1-4% DMSO may help.6
Multiple products Too many cycles Nonspecific bands may be eliminated with reduced cycling.
Annealing temperature too low Increase the annealing/extension temperature in increments of 2-3 °C.
Sub-optimal primer design See recommendations under “No PCR product”
Contaminant DNA in PCR If reagents other than Sigma MTP reagents were used in PCR reactions, it is likely that those unqualified reagents contained contaminating DNA. Otherwise, if Sigma MTP reagents were used, check your methodology.  All PCR reactions should be assembled in a hood in a clean area that is free from PCR products. Clean areas should be wiped down with 10% bleach after each use. All reagents should be portioned into small aliquots upon receipt to minimize the risk of contamination. Once contaminated, all suspect reagents should be discarded, and PCR repeated with fresh reagents.
Products are smeared Too many cycles Reduce the cycle number in 3-5 cycle increments.
Denaturation temperature too low Increase the denaturation temperature in 1 °C increments.
Extension time too long Decrease the extension time in 1-2 minute increments
Multiple PCR products generated Perform Touchdown PCR.8
Too much enzyme 0.5 µL (2.5 units) is sufficient for most applications. However, this concentration may be too high for some applications. It is recommended the cycling parameters be optimized first, as described above, then, reduce the enzyme concentration to 0.5‑0.2x.
[Mg++] too high In general magnesium should be 0.7 mM above the dNTP concentration.  Some reactions may be benefited by higher concentrations. Titrate Mg+2 in 0.2 mM increments.5
Template concentration too high Reduce template concentration in a series of 10 fold dilutions.
Product is wrong size Sub-optimal primer design See recommendations under “No PCR product”
Extension time too short Increase the extension times or use touchdown PCR.8
Contaminant DNA in PCR See recommendations under “Multiple products”
Faint Product [Template] too low Add additional template in 50 ng increments for genomic or 1‑2 ng for viral or plasmid DNA
Insufficient PCR cycles Increase the cycle number in 3-5 cycle increments
Extension time too short Increase the extension times in 2 minute increments
Sub-optimum reaction conditions Add PCR enhancers, e.g. 1-4% dimethyl sulfoxide (DMSO), D9170 or 0.8-1.3 M betaine B0300, final concentration.5

Materials

     

JumpStart is a trademark of Sigma-Aldrich Co. LLC
MTP is a trademark of Sigma-Aldrich Co. LLC
Restorase is a registered trademark of Sigma-Aldrich Co. LLC
TWEEN is a registered trademark of Croda International PLC

NOTICE TO PURCHASER: DISCLAIMER OF LICENSE

No license is conveyed with the purchase of this product under any of US Patents Nos. 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5’ Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

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