Mar 14, 2018
How to setup and perform a PCR experiment using GoTaq® G2 Master Mixes
- Promega Corporation1
- 1Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711
- Promega
External link: https://www.promega.com/-/media/files/resources/protocols/product-information-sheets/g/gotaq-g2-green-master-mix-protocol.pdf?la=en
Protocol Citation: Promega Corporation 2018. How to setup and perform a PCR experiment using GoTaq® G2 Master Mixes. protocols.io https://dx.doi.org/10.17504/protocols.io.k5icy4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 05, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 9098
Keywords: dna templates by pcr, pcr experiment, agarose gel electrophoresi, dna concentration, dna template, gel electrophoresi, polymerase, electrophoresi, pcr, dna, fluorescent gel scanner, mgcl2, blue dyes in the reaction buffer, fluorescence detection instrumentation, agarose gel, exonuclease activity, fluorescence excitation
Abstract
GoTaq® G2 Green Master Mix(a,b) is a premixed ready-to-use solution containing GoTaq® G2 DNA Polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of a wide range of DNA templates by PCR. GoTaq® G2 Green Master Mix contains two dyes (blue and yellow) that allow monitoring of progress during electrophoresis. Reactions assembled with GoTaq® G2 Green Master Mix have sufficient density for direct loading onto agarose gels. GoTaq® G2 DNA Polymerase exhibits 5 →3 exonuclease activity.
GoTaq® G2 Green Master Mix is recommended for any amplification reaction that will be visualized by agarose gel electrophoresis and ethidium bromide staining. The master mix is not recommended if any downstream applications use absorbance or fluorescence excitation, as the yellow and blue dyes in the reaction buffer may interfere with these applications. The dyes absorb wavelengths in the range of 225–300nm, making standard A260 readings to determine DNA concentration unreliable. The dyes have excitation peaks at 488nm and in the range of 600–700nm that correspond to the excitation wavelengths commonly used in fluorescence detection instrumentation. For some instrumentation, such as a fluorescent gel scanner that uses a 488nm excitation wavelength, there will be minimal interference since it is the yellow dye that absorbs at this wavelength. Gels scanned by this method will have a light grey dye front (corresponding to the yellow dye front) below the primers.
Attachments
Guidelines
General Guidelines for Amplification by PCR
A. Denaturation
- Generally, a 2-minute initial denaturation step at 95°C is sufficient.
- Subsequent denaturation steps will be between 15 seconds and 1 minute.
B. Annealing
- Optimize the annealing conditions by performing the reaction starting approximately 5°C below the calculated melting temperature of the primers and increasing the temperature in increments of 1°C to the annealing temperature.
- The annealing step is typically 15 seconds to 1 minute.
C. Extension
- The extension reaction is typically performed at the optimal temperature for Taq DNA polymerase, which is 72–74°C.
- Allow approximately 1 minute for every 1kb of DNA to be amplified.
- A final extension of 5 minutes at 72–74°C is recommended.
D. Refrigeration
- If the thermal cycler has a refrigeration or ”soak” cycle, the cycling reaction can be programmed to end by holding the tubes at 4°C for several hours.
- This cycle can minimize any polymerase activity that might occur at higher temperatures, although this is not usually a problem.
E. Cycle Number
- Generally, 25–30 cycles result in optimal amplification of desired products.
- Occasionally, up to 40 cycles may be performed, especially for detection of low-copy targets.
General Considerations
A. GoTaq® G2 Green Master Mix Compatibility
GoTaq® G2 Green Master Mix is compatible with common PCR additives such as DMSO and betaine. These additives neither change the color of GoTaq® G2 Green Master Mix nor affect dye migration.
If both agarose gel analysis and further downstream applications involving absorbance or fluorescence will be used, the two dyes can be removed from reactions using standard PCR clean-up systems such as the Wizard® SV Gel and PCR Clean-Up System (Cat.#A9281).
B. Primer Design
PCR primers generally range in length from 15–30 bases and are designed to flank the region of interest. Primers should contain 40–60% (G + C), and care should be taken to avoid sequences that might produce internal secondary structure. The 3 -ends of the primers should not be complementary to avoid the production of primer-dimers. Primerdimers unnecessarily deplete primers from the reaction and result in an unwanted polymerase reaction that competes with the desired reaction. Avoid three G or C nucleotides in a row near the 3 -end of the primer, as this may result in nonspecific primer annealing, increasing the synthesis of undesirable reaction products. Ideally, both primers should have nearly identical melting temperatures (Tm); in this manner, the two primers should anneal roughly at the same temperature. The annealing temperature of the reaction is dependent upon the primer with the lowest Tm. For assistance with calculating the Tm of any primer, a Tm Calculator is provided on the BioMath page of the Promega web site at: www.promega.com/biomath/
C. Amplification Troubleshooting
To overcome low yield or no yield in amplifications (e.g., mouse tail genotyping applications), we recommend the following suggestions:
- Adjust annealing temperature. The reaction buffer composition affects the melting properties of DNA. See BioMath Calculator to calculate the melting temperature for primers in the GoTaq® reaction (www.promega.com/biomath/).
- Minimize the effect of amplification inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the copurification of amplification inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
- Increase template DNA purity. Include an ethanol precipitation and wash step prior to amplification to remove inhibitors that copurify with the DNA.
- Add PCR additives. Adding PCR-enhancing agents (e.g., DMSO or betaine) may improve yields. General stabilizing agents such as BSA (Sigma Cat.# A7030; final concentration 0.16mg/ml) also may help to overcome amplification failure.
D. More Information on Amplification
More information on amplification is available online at the Promega web site: www.promega.com/products/pcr/
Materials
MATERIALS
GoTaq® G2 Green Master MixPromegaCatalog # M7822
template DNA
upstream primer
downstream primer
mineral oil (optional)
STEP MATERIALS
GoTaq® G2 Green Master MixPromegaCatalog #M7822
GoTaq® G2 Green Master MixPromegaCatalog #M7822
Protocol materials
GoTaq® G2 Green Master MixPromegaCatalog #M7822
GoTaq® G2 Green Master MixPromegaCatalog # M7822
template DNA
upstream primer
downstream primer
mineral oil (optional)
Troubleshooting
Thaw the GoTaq® G2 Green Master Mix at room temperature.
GoTaq® G2 Green Master MixPromegaCatalog #M7822
20 °C Thawing
Note
Minimize the number of freezethaw cycles by storing in working aliquots.
Product may be stored at 4°C for up to 18 weeks.
Mix well prior to use.
Vortex the Master Mix, then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube.
Prepare one of the following reaction mixes on ice:
Note
If using a thermal cycler without a heated lid, overlay the reaction mix with 1–2 drops (approximately 50μl) of mineral oil to prevent evaporation during thermal cycling.
50 µL Mineral oil
Centrifuge the reactions in a microcentrifuge for 5 seconds.
00:00:05 Centrifugation
Place the reactions in a thermal cycler that has been preheated to 95°C.
95 °C Preheated thermal cycler
Perform PCR using your standard parameters.