DTI CobaltAmp HiFid Taq HS Premix is a Fast PCR Master Mix and designed for streamlined PCR, and is, therefore, ideal for applications such as colony PCR. It contains a hot-start PCR enzyme, optimized buffer, dNTPs, along with a blue gel-loading dye and density reagent, in a convenient 2X premix format.
| Catalog # | Pack Size |
|---|---|
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DT0203.80 |
80 rxn (25 μl volume) |
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DT0203.320 |
320 rxn (25 μl volume) |
DTI CobaltAmp HiFid Taq HS Premix is a Fast PCR Master Mix and designed for streamlined PCR, and is, therefore, ideal for applications such as colony PCR. It contains a hot-start PCR enzyme, optimized buffer, dNTPs, along with a blue gel-loading dye and density reagent, in a convenient 2X premix format. Reactions are assembled by simply mixing primers and DNA template with DTI CobaltAmp HiFid Taq HS Premix. For rapid and convenient screening, the amplification reactions can be loaded directly onto an agarose gel immediately after amplification without purification, or digested directly with restriction enzymes.
Hot-Start Optimization
Hot-start PCR master mix is optimized for fast PCR.
Colony PCR Efficiency
Colony PCR can be completed in half the time required for conventional Taq DNA polymerase.
Convenient Protocol
Convenient protocol eliminates purification steps with no reduction in yield.
Direct Enzyme Digestion
Restriction enzyme digestion of the PCR products can be performed directly in the PCR buffer.
Gel-Ready Reactions
Reactions can be directly loaded onto a gel without purification or the addition of other reagents.
| Document | Download Link |
|---|---|
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DTI CobaltAmp HiFid Taq HS Premix – Protocol |
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DTI CobaltAmp HiFid Taq HS Premix – Certificate of Analysis (80 rxn) |
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DTI CobaltAmp HiFid Taq HS Premix – Certificate of Analysis (320 rxn) |
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DTI CobaltAmp HiFid Taq HS Premix – Promotional Document |
| Amplicon Length | DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix | Company P Dye-Added Master Mix | Company T Dye-Added Master Mix |
|---|---|---|---|
|
0.5 kb |
50 min |
1 hr 35 min |
1 hr 30 min |
|
1.0 kb |
55 min |
1 hr 50 min |
1 hr 45 min |
|
2.0 kb |
1 hr |
2 hr 20 min |
2 hr 15 min |
|
4.0 kb |
1 hr 30 min |
3 hr 20 min |
3 hr 15 min |
Experimental example: Using DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix to screen a cDNA library by colony PCR
E. coli cells were transformed with a mouse cDNA library (insert size from 0.5 to 5 kb) ligated into pUC118. Plasmid-containing transformants were analyzed using DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix and M4 and RV primers (see Methods below). The PCR reaction cycle was complete in just 70 minutes, and excellent yields were obtained. All 16 clones checked were confirmed to contain 0.5- to 5-kb inserts.
Figure 1: Colony PCR with DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix.
Methods
PCR master mix was prepared by including the following components (per reaction): DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix (2X Premix), 25 µl; M13 Primer M4 (20 µM), 0.5 µl; M13 Primer RV (20 µM), 0.5 µl; dH2O, 24 µl. PCR master mix aliquots (50 µl) were dispensed into each PCR tube. A sterile micropipette tip was used to transfer a few cells from each colony to a corresponding PCR tube, where cells were resuspended in PCR master mix. PCR was performed using a DTI PCR Thermal Cycler Dice thermal cycler (not available in all geographic locations). The cycling conditions were: 94°C, 1 min; followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec. After PCR was complete, 5 µl of each reaction was subjected to electrophoresis on a 1% L03 agarose gel.
We recommend DTI JadeAmp FabTaq Premix (Cat.# DT0201.320). This product is completely premixed, making it easy to prepare PCR reaction mixtures which can be loaded directly on an agarose gel for electrophoresis after the reaction. For an efficient workflow with minimal pipetting, DTI JadeAmp FabTaq Premix contains a green tracking dye. Additionally, a density agent is included to aid in gel loading.
DTI JadeAmp FabTaq Premix can amplify targets up to 10 kb in length, including targets that are GC- or AT-rich. Additionally, PCR products generated with DTI JadeAmp FabTaq Premix can be used directly for restriction enzyme digestion, sequencing, or TA-cloning without the need for further purification.
We recommend DTI CobaltAmp HiFid Taq HS Premix for colony PCR. These enzyme preparations tolerate the presence of substantial bacterial nucleic acid carry-over. Because tracking dye and density agent are included in these master mixes, the PCR reaction mixtures may be loaded directly on an agarose gel for electrophoresis.
DTI CobaltAmp HiFid Taq HS Premix is an economical choice for high-throughput projects. This enzyme is formulated to include high-speed polymerase, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent in a 2X premix. Since it requires an extension time of only 10 sec/kb, colony PCR reactions can be completed in less than 1 hour for inserts up to 1 kb.
DTI FabTaq and DTI FabTaq Hot Start Version enzymes are compatible with inosine-containing primers. It is important to note that inosine-containing primers should not be used with PCR enzymes that have 3’→5’ exonuclease activity. When using one of these PCR enzymes, we recommend using mixtures of degenerate primers with A, T, G, and C at the desired position(s) rather than inosine-containing primers when performing degenerate PCR.
The optimal amount of template required depends on the complexity of the template and the copy number of the target sequence. Approximately 10⁴ copies of the target DNA sequence are required to detect the amplification product in 25–30 PCR cycles.
• Typically, 1 µg of human genomic DNA contains 3.04 x 10⁵ molecules of DNA. For most PCR applications, 30–100 ng of human genomic DNA is sufficient. High-copy targets, such as housekeeping genes, require only 10 ng of template. Template amounts for higher-complexity templates range between 10 ng and 500 ng.
• Typically, 1 µg of E. coli genomic DNA contains 2 x 10⁸ molecules of DNA; therefore, the recommended amount of template is between 100 pg and 1 ng.
• Typically, 1 µg of lambda DNA contains 1.9 x 10¹⁰ molecules of DNA; therefore, the template input can be as little as 100 pg.
• The amount of cDNA template depends on the copy number of the target. cDNA input is typically described in terms of equivalent RNA input. The amount of cDNA in a PCR reaction can be as little as 10 pg (RNA equivalent).
For some polymerases, excessive template tends to inhibit amplification. In contrast, some DNA polymerases tolerate a broader range of template quantity. In general, reaction conditions may be adjusted depending on the type, quantity, and quality of the template.