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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.

Specifications

Documents

Applications

FAQs

Brand	Catalogue Number	Pack Size
DTI CobaltAmp HiFid Taq HS Premix	DT0203.80	80 rxn (25 μl volume)
DTI CobaltAmp HiFid Taq HS Premix	DT0203.320	320 rxn (25 μl volume)

• Hot-start PCR master mix is optimized for fast PCR.

• Colony PCR can be completed in half the time required for conventional Taq DNA polymerase.

• Convenient protocol eliminates purification steps with no reduction in yield.

• Restriction enzyme digestion of the PCR products can be performed directly in the PCR buffer.

• Reactions can be directly loaded onto a gel without purification or the addition of other reagents.

DTI CobaltAmp HiFid Taq HS Premix - Protocol

DTI CobaltAmp HiFid Taq HS Premix - Certificate of Analysis

DTI CobaltAmp HiFid Taq HS Premix - Promotional Document

Results

Speed up Colony PCR with DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix

DTI CobaltAmp HiFid Taq HS Premix PCR Master Mix is fast—with an extension speed of 10 seconds/kb, reactions can be completed in half the time of conventional Taq. CobaltAmp master mix is also significantly faster than dye-added premixes offered by other manufacturers. The table below lists reaction times for CobaltAmp master mix and the recommended reaction times for two other commercially available dye-added premixes.

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 2. 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.

Which enzymes faciliate efficient workflow when analyzing multiple samples by gel electrophoresis after PCR (e.g., genotyping screens)?

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, 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 need for further purification.

Which enzymes are suitable for colony PCR?

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.

Which enzymes are suitable for fast PCR?

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.

What precautions should be taken when using an inosine-containing primer?

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.

What is the terminal structure of DTI FabTaq and DTI FabTaq Hot Start Version enzymes’ amplification product?

DTI FabTaq and DTI FabTaq Hot Start Version enzymes primarily yield amplification products containing 3’-dA overhangs, which facilitates TA cloning.

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