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Platinum Direct PCR Universal Master MixRequest sample |
Direct PCR: Universal master mix for a direct route to PCR
Direct PCR allows DNA sequence amplification directly from samples, without extracting and purifying the DNA. Invitrogen Platinum Direct PCR Universal Master Mix is an excellent choice for amplification of target DNA from crude samples in applications such as mouse genotyping, CRISPR-edited cell analysis, and target DNA detection from limited materials.
Watch the video on what is direct PCR, how it works, and how to get the most out of a direct PCR kit.
Click image to enlarge
Figure 1. Comparison of traditional PCR to direct PCR.
Highlights of Platinum Direct PCR Universal Master Mix
The master mix is formulated with Invitrogen Platinum II Taq Hot-Start DNA Polymerase, which is engineered for high inhibitor tolerance, fast DNA synthesis, and high sensitivity. In addition to the enzyme, the master mix consists of buffer, dNTPs, and other reaction components, and a green dye for direct gel loading. The Lysis Buffer, Proteinase K, and the Invitrogen Platinum GC Enhancer are also included as separate vials in the package to enable full workflow. Platinum Direct PCR Master Mix provides two protocols to amplify target DNA from crude samples (Figure 1). In the direct protocol, a sample is directly added to the master mix for amplification. In the lysis protocol, the sample is first lysed and then amplified. Samples can be stored in the lysis buffer for up to 12 months.
Benefits include:
- Universal—One master mix for different sample types, one annealing temperature for different primer sets
- Convenient—Buffer formulated for primer annealing at 60°C, helping simplify PCR optimization
- Fast—No DNA purification; DNA synthesis at 20 sec/kb
- Efficient—High inhibitor tolerance; GC enhancer included for amplification of GC-rich sequences
- Flexible—Optional lysis-and-storage protocol for long-term storage and further testing
- Fewer pipetting—Master mix format with direct gel-loading dye
Platinum Direct PCR Universal Master Mix reduces the use of hazardous reagents and generates 99% less plastic waste than traditional DNA extraction workflows using spin columns.
Read green fact sheet on Platinum Direct PCR Universal Master Mix
Samples tested with Platinum Direct PCR Universal Master Mix
Direct PCR from human samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various types of human tissues—saliva, buccal swab, blood (preserved in citrate, heparin, or EDTA), nail, hair, and HeLa S3 cells.
Click image to enlarge
Figure 2. Direct DNA amplification from various human research tissues. 0.35 kb and 7.5 kb fragments were amplified from various types of human tissues with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is Invitrogen TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from mouse samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various types of mouse tissues—ear, tail, kidney, heart, pancreas, liver, spleen, brain, hair, bone marrow, bone, and tooth.
Click image to enlarge
Figure 3. Direct DNA amplification from various mouse tissues. 0.3 kb and 3.6 kb fragments were amplified from various types of mouse tissues with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. Soft tissue was cut into 1 mm pieces, hard tissue like bone was crushed in a ceramic grinder to 1–2 mm. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Read article: Mouse-genotyping PCR tips
Direct PCR from plant tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from plant tissues of various species—wheat leaf, tobacco leaf, birch leaf, maple leaf, magnolia leaf, orchid leaf, Arabidopsis leaf, rose leaf, linden leaf, lilac leaf, strawberry leaf, sild strawberry leaf, Thuja leaf, pine spike, spruce spike, rosehips petal, strawberry, corn seed, peanut, wheat seed, tobacco seed, sunflower seed, and wheat root.
Click image to enlarge
Figure 4. Direct DNA amplification from different plant species and tissue types. A 0.3 kb fragment was amplified directly from leaves, seeds, roots, and fruits of plant species listed with the Platinum Direct PCR Universal Master Mix. The direct protocol was followed for the results shown by using 1 mm leaf punch or crushed seed. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from algae
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from algae with or without cell wall.
Click image to enlarge
Figure 5. Direct DNA amplification from algae. A0.11 kb fragment was amplified directly from the single-cell green algae Chlamydomonas reinhardtii with and without the cell wall using the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from bird tissue samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various bird tissues—chicken cartilage, chicken meat, and quail feather.
Click image to enlarge
Figure 6. Direct DNA amplification from bird tissue. A 0.24 kb fragment was amplified directly from chicken (cartilage, meat) and quail (feather) with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from bacteria
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from gram-positive and gram-negative bacterial species (Figure 7) and can be used in bacterial DNA detection.
Click image to enlarge
Figure 7. Direct DNA amplification from bacteria. 0.62 kb and 0.59 kb fragments were amplified directly from the gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria respectively with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Click image to enlarge
Figure 8. Direct DNA amplification for bacterial detection. A 0.45 kb fragment of bacterial 16S rRNA sequence was amplified directly from surface swabs of fruits and vegetables (U = unwashed, W = washed), using the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is Thermo Scientific GeneRuler 1 kb DNA Ladder. NTC = no-template control (swab without samples).
Direct PCR from zebrafish tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various zebrafish tissues—fin, scale, muscle, and brain.
Click image to enlarge
Figure 9. Direct DNA amplification from zebrafish. 0.35 kb and 1 kb fragments were amplified directly from zebrafish tissues with the Platinum Direct PCR Universal Master Mix. Results from the lysis protocol are shown. Successful amplification was observed with fin, scale, muscle, and brain of the zebrafish. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from Drosophila tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various Drosophila tissues—whole body, half body, head, and half larva.
Click image to enlarge
Figure 10. Direct DNA amplification from Drosophila. A 0.66 kb fragment was amplified directly from various Drosophila tissues with the Platinum Direct PCR Universal Master Mix. Results from the lysis protocol are shown. Successful amplification was observed with body, head, and larva of Drosophila. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from blood
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from human blood preserved in citrate, heparin, or EDTA.
Figure 11. Direct DNA amplification from human blood research samples. 0.35 kb and 7.5 kb fragments were amplified from human blood preserved in citrate, heparin, or EDTA with Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from mouse FFPE
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from a mouse kidney FFPE sample.
Click image to enlarge
Figure 12. Direct DNA amplification from FFPE samples. Fragments of different lengths (0.1 kb, 0.2 kb, 0.3 kb, 0.5 kb, 1 kb) were amplified from mouse kidney FFPE blocks with the Platinum Direct PCR Universal Master Mix according to the manual recommendations. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder. Note that FFPE DNA is often fragmented, and sample quality may not allow amplification longer than 0.3 kb.
Direct PCR from human samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various types of human tissues—saliva, buccal swab, blood (preserved in citrate, heparin, or EDTA), nail, hair, and HeLa S3 cells.
Click image to enlarge
Figure 2. Direct DNA amplification from various human research tissues. 0.35 kb and 7.5 kb fragments were amplified from various types of human tissues with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is Invitrogen TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from mouse samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various types of mouse tissues—ear, tail, kidney, heart, pancreas, liver, spleen, brain, hair, bone marrow, bone, and tooth.
Click image to enlarge
Figure 3. Direct DNA amplification from various mouse tissues. 0.3 kb and 3.6 kb fragments were amplified from various types of mouse tissues with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. Soft tissue was cut into 1 mm pieces, hard tissue like bone was crushed in a ceramic grinder to 1–2 mm. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Read article: Mouse-genotyping PCR tips
Direct PCR from plant tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from plant tissues of various species—wheat leaf, tobacco leaf, birch leaf, maple leaf, magnolia leaf, orchid leaf, Arabidopsis leaf, rose leaf, linden leaf, lilac leaf, strawberry leaf, sild strawberry leaf, Thuja leaf, pine spike, spruce spike, rosehips petal, strawberry, corn seed, peanut, wheat seed, tobacco seed, sunflower seed, and wheat root.
Click image to enlarge
Figure 4. Direct DNA amplification from different plant species and tissue types. A 0.3 kb fragment was amplified directly from leaves, seeds, roots, and fruits of plant species listed with the Platinum Direct PCR Universal Master Mix. The direct protocol was followed for the results shown by using 1 mm leaf punch or crushed seed. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from algae
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from algae with or without cell wall.
Click image to enlarge
Figure 5. Direct DNA amplification from algae. A0.11 kb fragment was amplified directly from the single-cell green algae Chlamydomonas reinhardtii with and without the cell wall using the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from bird tissue samples
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various bird tissues—chicken cartilage, chicken meat, and quail feather.
Click image to enlarge
Figure 6. Direct DNA amplification from bird tissue. A 0.24 kb fragment was amplified directly from chicken (cartilage, meat) and quail (feather) with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from bacteria
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from gram-positive and gram-negative bacterial species (Figure 7) and can be used in bacterial DNA detection.
Click image to enlarge
Figure 7. Direct DNA amplification from bacteria. 0.62 kb and 0.59 kb fragments were amplified directly from the gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria respectively with the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Click image to enlarge
Figure 8. Direct DNA amplification for bacterial detection. A 0.45 kb fragment of bacterial 16S rRNA sequence was amplified directly from surface swabs of fruits and vegetables (U = unwashed, W = washed), using the Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is Thermo Scientific GeneRuler 1 kb DNA Ladder. NTC = no-template control (swab without samples).
Direct PCR from zebrafish tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various zebrafish tissues—fin, scale, muscle, and brain.
Click image to enlarge
Figure 9. Direct DNA amplification from zebrafish. 0.35 kb and 1 kb fragments were amplified directly from zebrafish tissues with the Platinum Direct PCR Universal Master Mix. Results from the lysis protocol are shown. Successful amplification was observed with fin, scale, muscle, and brain of the zebrafish. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from Drosophila tissue
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from various Drosophila tissues—whole body, half body, head, and half larva.
Click image to enlarge
Figure 10. Direct DNA amplification from Drosophila. A 0.66 kb fragment was amplified directly from various Drosophila tissues with the Platinum Direct PCR Universal Master Mix. Results from the lysis protocol are shown. Successful amplification was observed with body, head, and larva of Drosophila. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from blood
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from human blood preserved in citrate, heparin, or EDTA.
Figure 11. Direct DNA amplification from human blood research samples. 0.35 kb and 7.5 kb fragments were amplified from human blood preserved in citrate, heparin, or EDTA with Platinum Direct PCR Universal Master Mix. The lysis protocol was followed for the results shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Direct PCR from mouse FFPE
Platinum Direct PCR Universal Master Mix successfully amplifies target DNA sequences from a mouse kidney FFPE sample.
Click image to enlarge
Figure 12. Direct DNA amplification from FFPE samples. Fragments of different lengths (0.1 kb, 0.2 kb, 0.3 kb, 0.5 kb, 1 kb) were amplified from mouse kidney FFPE blocks with the Platinum Direct PCR Universal Master Mix according to the manual recommendations. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder. Note that FFPE DNA is often fragmented, and sample quality may not allow amplification longer than 0.3 kb.
Benefits of Platinum Direct PCR Universal Master Mix
The innovative Platinum direct PCR master mix's buffer formulation enables annealing of primers at 60°C (universal annealing temperature) regardless of their sequences that follow general primer design rules (Figure 13). The buffer also allows successful amplification when calculated Tms are used in the annealing step (data not shown). This unique buffer also allows assay co-cycling (Figure 14) and cycling of longer and shorter amplicons together.
Click image to enlarge
Figure 13. Platinum Direct PCR Universal Master Mix produces PCR products with high specificity and yield when DNA templates of various samples were amplified using the universal annealing temperature at 60°C. Primer sets of varying annealing temperatures (indicated above) were used to amplify five targets from different tissues of plant, mouse, and human. Results of both direct and lysis protocols are shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Due to its innovative buffer, Platinum Direct PCR Universal Master Mix allows for a universal annealing temperature and flexible extension time for co-cycling of all assays.
Click image to enlarge
Figure 14. Time saving and assay co-cycling enabled by universal PCR protocol. With the Platinum Direct PCR Universal Master Mix, different PCR assays can be cycled together (co-cycling) using one protocol with a universal primer annealing temperature and the extension time selected for the longest fragment to be amplified (Figure 15).
Click image to enlarge
Figure 15. Platinum Direct PCR Universal Master Mix enables cycling of shorter and longer amplicons together. 0.2 kb, 0.6 kb, 1 kb, 1.5 kb, and 2 kb fragments were amplified from samples of different species and tissue types, using the same protocol for all five targets: 94°C denaturation for 15 sec, 60°C annealing for 15 sec, 68°C extension for 40 sec. The extension time was based on length of the longest target.
To learn more about universal protocol, view the Spotlight article: PCR simplified with universal primer annealing
Platinum Direct PCR Universal Master Mix is provided as a green master mix for direct gel loading of PCR products, eliminating tedious steps of dye addition to PCR samples and helping reduce pipetting errors and hands-on time.
Figure 16. The green master mix for loading PCR products directly to a gel for analysis. The Platinum Direct PCR Universal Master Mix contains a density reagent and two tracking dyes. DNA migration is easily tracked with two dyes (blue and yellow) that are readily visible during electrophoresis (the lanes for 5 and 15 min in the figure to the right).
Platinum Direct PCR Master Mix provides two protocols to amplify target DNA from crude samples. In the direct protocol, a sample of recommended size is added directly to the master mix. In the lysis protocol, a sample of recommended size is lysed first, then a fraction of its supernatant is transferred to the master mix. The direct protocol offers a shorter workflow whereas the lysis protocol allows for a flexible workflow with a sample-storage option (Figure 16). Up to 2 kb can be amplified using the direct PCR protocol, and up to 8 kb can be amplified following the lysis protocol. Samples can be stored in the lysis buffer for up to 12 months.
Sample size and amount are critical for success in direct PCR. Low sample amount can cause PCR failure due to insufficient input. On the other hand, large sample size introduces cellular components and debris that can inhibit PCR (Figure 17). Recommended sample sizes are provided in the manual of the Platinum Direct PCR Universal Master Mix.
Click image to enlarge
Figure 17. Direct protocol vs. lysis protocol.
Click image to enlarge
Figure 18. Sample size recommendation for the direct and lysis protocol (not rendered for actual size). In general, 0.5–1.0 mm of solid samples works well with the direct protocol. 0.5–2.0 mm is recommended for the lysis protocol although up to 10 mm of sample can work with the lysis protocol. Please refer to the product manual for detailed recommendations on different sample types.
The innovative Platinum direct PCR master mix's buffer formulation enables annealing of primers at 60°C (universal annealing temperature) regardless of their sequences that follow general primer design rules (Figure 13). The buffer also allows successful amplification when calculated Tms are used in the annealing step (data not shown). This unique buffer also allows assay co-cycling (Figure 14) and cycling of longer and shorter amplicons together.
Click image to enlarge
Figure 13. Platinum Direct PCR Universal Master Mix produces PCR products with high specificity and yield when DNA templates of various samples were amplified using the universal annealing temperature at 60°C. Primer sets of varying annealing temperatures (indicated above) were used to amplify five targets from different tissues of plant, mouse, and human. Results of both direct and lysis protocols are shown. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Due to its innovative buffer, Platinum Direct PCR Universal Master Mix allows for a universal annealing temperature and flexible extension time for co-cycling of all assays.
Click image to enlarge
Figure 14. Time saving and assay co-cycling enabled by universal PCR protocol. With the Platinum Direct PCR Universal Master Mix, different PCR assays can be cycled together (co-cycling) using one protocol with a universal primer annealing temperature and the extension time selected for the longest fragment to be amplified (Figure 15).
Click image to enlarge
Figure 15. Platinum Direct PCR Universal Master Mix enables cycling of shorter and longer amplicons together. 0.2 kb, 0.6 kb, 1 kb, 1.5 kb, and 2 kb fragments were amplified from samples of different species and tissue types, using the same protocol for all five targets: 94°C denaturation for 15 sec, 60°C annealing for 15 sec, 68°C extension for 40 sec. The extension time was based on length of the longest target.
To learn more about universal protocol, view the Spotlight article: PCR simplified with universal primer annealing
Platinum Direct PCR Universal Master Mix is provided as a green master mix for direct gel loading of PCR products, eliminating tedious steps of dye addition to PCR samples and helping reduce pipetting errors and hands-on time.
Figure 16. The green master mix for loading PCR products directly to a gel for analysis. The Platinum Direct PCR Universal Master Mix contains a density reagent and two tracking dyes. DNA migration is easily tracked with two dyes (blue and yellow) that are readily visible during electrophoresis (the lanes for 5 and 15 min in the figure to the right).
Platinum Direct PCR Master Mix provides two protocols to amplify target DNA from crude samples. In the direct protocol, a sample of recommended size is added directly to the master mix. In the lysis protocol, a sample of recommended size is lysed first, then a fraction of its supernatant is transferred to the master mix. The direct protocol offers a shorter workflow whereas the lysis protocol allows for a flexible workflow with a sample-storage option (Figure 16). Up to 2 kb can be amplified using the direct PCR protocol, and up to 8 kb can be amplified following the lysis protocol. Samples can be stored in the lysis buffer for up to 12 months.
Sample size and amount are critical for success in direct PCR. Low sample amount can cause PCR failure due to insufficient input. On the other hand, large sample size introduces cellular components and debris that can inhibit PCR (Figure 17). Recommended sample sizes are provided in the manual of the Platinum Direct PCR Universal Master Mix.
Click image to enlarge
Figure 17. Direct protocol vs. lysis protocol.
Click image to enlarge
Figure 18. Sample size recommendation for the direct and lysis protocol (not rendered for actual size). In general, 0.5–1.0 mm of solid samples works well with the direct protocol. 0.5–2.0 mm is recommended for the lysis protocol although up to 10 mm of sample can work with the lysis protocol. Please refer to the product manual for detailed recommendations on different sample types.
Improve PCR efficiency with Platinum Direct PCR Universal Master Mix
Platinum Direct PCR Universal Master Mix contains Platinum II Taq Hot-Start DNA Polymerase, a DNA polymerase engineered for fast DNA synthesis. Therefore, PCR results can be obtained sooner with the Platinum direct PCR master mix than other direct PCR enzyme kits (Figure 19).
Click image to enlarge
Figure 19. Fast cycling reduces PCR run time. A 1 kb fragment was amplified for 35 cycles using the Platinum Direct PCR Universal Master Mix and direct PCR enzyme kits from other suppliers. Cycling times for each kit are shown in purple, while ramping times on the Applied Biosystems ProFlex PCR System (6°C/sec peak block ramp rate) are shown in red.
Platinum Direct PCR Universal Master Mix enables amplification of long sequences directly from the samples.
Figure 20. Amplification ranges of the direct and lysis protocols using Platinum Direct PCR Universal Master Mix. Fragments up to 2 kb can be amplified with the direct protocol while fragments up to 8 kb can be amplified with the lysis protocol. Results shown are PCR with human blood preserved in heparin. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix contains Platinum II Taq Hot-Start DNA Polymerase and Platinum hot-start. This antibody-based hot-start technology leads to superior specificity in direct DNA amplification using crude samples.
Click image to enlarge
Figure 21. High specificity of direct PCR. 0.3 kb, 0.5 kb, 1.1 kb, 2.2 kb, 2.9 kb, 3.6 kb, and 5.5 kb targets were amplified from mouse ear following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mixcontains Platinum II Taq Hot-Start DNA Polymerase, which is an engineered enzyme with improved sensitivity. Therefore, target sequences can be detected from low sample input using the Platinum Direct PCR Universal Master Mix.
Click image to enlarge
Figure 22. High sensitivity of PCR from low-sample input. 0.4 kb and 2.2 kb targets were amplified from 5–500 of HeLa S3 cells following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix allows for amplification of versatile range of targets, from AT-rich to GC-rich. A separate vial of Platinum GC Enhancer is provided for specific and enhanced amplification of targets with high-GC content.
Click image to enlarge
Figure 23. Robust direct amplification of AT-rich and GC-rich targets. Fourteen targets of varying GC content were amplified from human buccal swabs following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The Platinum GC Enhancer (included with the kit) was used for targets over 65% GC content. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix allows amplification of multiple targets in a single reaction.
Click image to enlarge
Figure 24. Efficient multiplexing of up to five targets using direct PCR. Fragments of different lengths (0.1 kb to 1.1 kb) were amplified from mouse ear, tail, and hair in single-plex to 5-plex reactions. Results from the lysis protocol of the Platinum Direct PCR Universal Master Mix are shown. The molecular weight marker is the Invitrogen TrackIt 100 bp DNA Ladder.
Extended stability of the Platinum Direct PCR Universal Master Mix at room temperature enables high-throughput applications. The Platinum hot-start technology enables benchtop stability and ensures target amplification with high specificity.
Click image to enlarge
Figure 25. Assembled reactions with Platinum Direct PCR Universal Master Mix are stable at room temperature. A 2.2 kb fragment was amplified from mouse tail, following the lysis protocol of the Platinum Direct PCR Master Mix. Reactions were set up at room temperature (25°C) or on ice (4°C), then immediately run for PCR or were left at room temperature (25°C) for 8 hours before PCR. Even after 8 hours at room temperature, the Platinum Direct PCR Master Mix produces results with high specificity and yield. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix contains Platinum II Taq Hot-Start DNA Polymerase, a DNA polymerase engineered for fast DNA synthesis. Therefore, PCR results can be obtained sooner with the Platinum direct PCR master mix than other direct PCR enzyme kits (Figure 19).
Click image to enlarge
Figure 19. Fast cycling reduces PCR run time. A 1 kb fragment was amplified for 35 cycles using the Platinum Direct PCR Universal Master Mix and direct PCR enzyme kits from other suppliers. Cycling times for each kit are shown in purple, while ramping times on the Applied Biosystems ProFlex PCR System (6°C/sec peak block ramp rate) are shown in red.
Platinum Direct PCR Universal Master Mix enables amplification of long sequences directly from the samples.
Figure 20. Amplification ranges of the direct and lysis protocols using Platinum Direct PCR Universal Master Mix. Fragments up to 2 kb can be amplified with the direct protocol while fragments up to 8 kb can be amplified with the lysis protocol. Results shown are PCR with human blood preserved in heparin. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix contains Platinum II Taq Hot-Start DNA Polymerase and Platinum hot-start. This antibody-based hot-start technology leads to superior specificity in direct DNA amplification using crude samples.
Click image to enlarge
Figure 21. High specificity of direct PCR. 0.3 kb, 0.5 kb, 1.1 kb, 2.2 kb, 2.9 kb, 3.6 kb, and 5.5 kb targets were amplified from mouse ear following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mixcontains Platinum II Taq Hot-Start DNA Polymerase, which is an engineered enzyme with improved sensitivity. Therefore, target sequences can be detected from low sample input using the Platinum Direct PCR Universal Master Mix.
Click image to enlarge
Figure 22. High sensitivity of PCR from low-sample input. 0.4 kb and 2.2 kb targets were amplified from 5–500 of HeLa S3 cells following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix allows for amplification of versatile range of targets, from AT-rich to GC-rich. A separate vial of Platinum GC Enhancer is provided for specific and enhanced amplification of targets with high-GC content.
Click image to enlarge
Figure 23. Robust direct amplification of AT-rich and GC-rich targets. Fourteen targets of varying GC content were amplified from human buccal swabs following the lysis protocol of the Platinum Direct PCR Universal Master Mix. The Platinum GC Enhancer (included with the kit) was used for targets over 65% GC content. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Platinum Direct PCR Universal Master Mix allows amplification of multiple targets in a single reaction.
Click image to enlarge
Figure 24. Efficient multiplexing of up to five targets using direct PCR. Fragments of different lengths (0.1 kb to 1.1 kb) were amplified from mouse ear, tail, and hair in single-plex to 5-plex reactions. Results from the lysis protocol of the Platinum Direct PCR Universal Master Mix are shown. The molecular weight marker is the Invitrogen TrackIt 100 bp DNA Ladder.
Extended stability of the Platinum Direct PCR Universal Master Mix at room temperature enables high-throughput applications. The Platinum hot-start technology enables benchtop stability and ensures target amplification with high specificity.
Click image to enlarge
Figure 25. Assembled reactions with Platinum Direct PCR Universal Master Mix are stable at room temperature. A 2.2 kb fragment was amplified from mouse tail, following the lysis protocol of the Platinum Direct PCR Master Mix. Reactions were set up at room temperature (25°C) or on ice (4°C), then immediately run for PCR or were left at room temperature (25°C) for 8 hours before PCR. Even after 8 hours at room temperature, the Platinum Direct PCR Master Mix produces results with high specificity and yield. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Benchmarking data with Direct PCR kits
Tobacco leaf samples (Figure 26) and tobacco seed samples (Figure 27) were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 26. Robust direct DNA amplification from tobacco leaves. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (of 0.15 kb, 0.6 kb, 0.75 kb, 1.6 kb, and 2.2 kb) from tobacco leaves, following the direct protocol. The same targets were also amplified using competitor direct PCR kits: (A) Bioline MyTaq Plant PCR Kit, (B) TaKaRa Terra PCR Direct Polymerase Mix, and (C) Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (lysis protocol). The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Click image to enlarge
Figure 27. Robust direct DNA amplification from tobacco seeds. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (0.15 kb, 0.6 kb, 1.6 kb, and 2.2 kb) from tobacco seeds, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit, (B) Bioline MyTaq Plant PCR Kit, and (C) Roche KAPA3G Plant PCR Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Mouse ear samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 28. Robust direct DNA amplification from mouse ear. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (0.3 kb, 0.5 kb, 1.1 kb, 2.2 kb, 2.9 kb, 3.6 kb, and 5.5 kb) from mouse ear punches, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma REDExtract-N-Amp Tissue PCR Kit, (B) Bioline MyTaq Extract PCR Kit, (C) TaKaRa Terra PCR Direct Polymerase Mix, and (D) Roche KAPA Mouse Genotyping Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Human saliva samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Figure 29. Robust direct DNA amplification from human saliva. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.8 kb, 2 kb, 4.5 kb, and 7.5 kb) from human saliva samples, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma-Aldrich REDExtract-N-Amp Tissue PCR Kit, (B) Bioline MyTaq Extract PCR Kit, and (C) TaKaRa Terra PCR Direct Polymerase Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Human blood research samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 30. Robust direct DNA amplification from human blood. Platinum Direct PCR Universal Master Mix (left panels) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.35 kb, 0.8 kb, 2 kb, 3.3 kb, 4.5 kb, and 7.5 kb) from human blood research samples, following the direct or lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Bioline™ MyTaq Blood PCR kit (direct protocol) and (B) TaKaRa™ Terra PCR Direct Polymerase Mix (lysis protocol).The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
A sample of human cell line was used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Figure 31. Robust direct DNA amplification from human cells. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.8 kb, 2 kb, 4.5 kb, and 7.5 kb) from HeLa S3 cells, following the direct or lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) TaKaRa™ Terra PCR Direct Polymerase Mix, (B) Sigma-Aldrich™ REDExtract-N-Amp Tissue PCR Kit, and (C) Bioline™ MyTaq Extract PCR Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Application note: Direct qPCR with cell samples
Mouse kidney FFPE samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 32. Robust in direct DNA amplification from FFPE blocks. Platinum Direct PCR Universal Master Mix (left panel) provides high specificity and yield across a range of DNA fragments (0.1 kb, 0.2 kb, 0.3 kb, 0.5 kb, and 1 kb) from mouse kidney FFPE slides, following the lysis protocol. The same targets were also amplified using TaKaRa™ Terra PCR Direct FFPE Kit (A). NTC stands for no-template control, and the molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder. Note that FFPE DNA is often fragmented, and sample quality may not allow amplification longer than 0.3 kb.
Tobacco leaf samples (Figure 26) and tobacco seed samples (Figure 27) were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 26. Robust direct DNA amplification from tobacco leaves. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (of 0.15 kb, 0.6 kb, 0.75 kb, 1.6 kb, and 2.2 kb) from tobacco leaves, following the direct protocol. The same targets were also amplified using competitor direct PCR kits: (A) Bioline MyTaq Plant PCR Kit, (B) TaKaRa Terra PCR Direct Polymerase Mix, and (C) Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (lysis protocol). The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Click image to enlarge
Figure 27. Robust direct DNA amplification from tobacco seeds. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (0.15 kb, 0.6 kb, 1.6 kb, and 2.2 kb) from tobacco seeds, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit, (B) Bioline MyTaq Plant PCR Kit, and (C) Roche KAPA3G Plant PCR Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Mouse ear samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 28. Robust direct DNA amplification from mouse ear. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across range of DNA fragments (0.3 kb, 0.5 kb, 1.1 kb, 2.2 kb, 2.9 kb, 3.6 kb, and 5.5 kb) from mouse ear punches, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma REDExtract-N-Amp Tissue PCR Kit, (B) Bioline MyTaq Extract PCR Kit, (C) TaKaRa Terra PCR Direct Polymerase Mix, and (D) Roche KAPA Mouse Genotyping Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Human saliva samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Figure 29. Robust direct DNA amplification from human saliva. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.8 kb, 2 kb, 4.5 kb, and 7.5 kb) from human saliva samples, following the lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Sigma-Aldrich REDExtract-N-Amp Tissue PCR Kit, (B) Bioline MyTaq Extract PCR Kit, and (C) TaKaRa Terra PCR Direct Polymerase Mix. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Human blood research samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 30. Robust direct DNA amplification from human blood. Platinum Direct PCR Universal Master Mix (left panels) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.35 kb, 0.8 kb, 2 kb, 3.3 kb, 4.5 kb, and 7.5 kb) from human blood research samples, following the direct or lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) Bioline™ MyTaq Blood PCR kit (direct protocol) and (B) TaKaRa™ Terra PCR Direct Polymerase Mix (lysis protocol).The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
A sample of human cell line was used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Figure 31. Robust direct DNA amplification from human cells. Platinum Direct PCR Universal Master Mix (far left panel) provides high specificity and yield across a range of DNA fragments (0.2 kb, 0.8 kb, 2 kb, 4.5 kb, and 7.5 kb) from HeLa S3 cells, following the direct or lysis protocol. The same targets were also amplified using competitor direct PCR kits: (A) TaKaRa™ Terra PCR Direct Polymerase Mix, (B) Sigma-Aldrich™ REDExtract-N-Amp Tissue PCR Kit, and (C) Bioline™ MyTaq Extract PCR Kit. The molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder.
Application note: Direct qPCR with cell samples
Mouse kidney FFPE samples were used with commercially available direct PCR kits to compare yield and specificity of target amplification.
Click image to enlarge
Figure 32. Robust in direct DNA amplification from FFPE blocks. Platinum Direct PCR Universal Master Mix (left panel) provides high specificity and yield across a range of DNA fragments (0.1 kb, 0.2 kb, 0.3 kb, 0.5 kb, and 1 kb) from mouse kidney FFPE slides, following the lysis protocol. The same targets were also amplified using TaKaRa™ Terra PCR Direct FFPE Kit (A). NTC stands for no-template control, and the molecular weight marker (M) is TrackIt 1 Kb Plus DNA Ladder. Note that FFPE DNA is often fragmented, and sample quality may not allow amplification longer than 0.3 kb.
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Stylesheet for Classic Wide Template adjustments
For Research Use Only. Not for use in diagnostic procedures.