Rai™ Taq DNA Polymerase

Cat. No.	Component	Quantity	Storage	Concentration
MCE0201-S (500 unit)	Rai Taq DNA Polymerase	1 x 0.10 ml	-20˚C	5,000 U/ml
	UTaq Buffer	1 x 1.5 ml	-20˚C	10X
	25 mM MgCl2	1 x 1.5 ml	25˚C	–
MCE0201-L (2000 unit)	Rai Taq DNA Polymerase	1 x 0.40 ml	-20˚C	5,000 U/ml
	UTaq Buffer	2 x 1.5 ml	-20˚C	10X
	25 mM MgCl2	2 x 1.5 ml	25˚C	–

To ensure consistency across parallel PCR reactions and minimize pipetting errors, it is recommended to prepare a PCR master mix. This mix should include nuclease-free water, PCR buffer, dNTPs, primers, and Taq DNA Polymerase. Always prepare slightly more than needed to account for pipetting losses—typically, one extra reaction volume.

Once prepared, aliquot the master mix into individual PCR tubes and add the DNA template separately.

Thawing: Thaw all reagents on ice. Gently vortex and briefly centrifuge to ensure homogeneity.

Reaction Assembly: Place thin-walled PCR tubes on ice. For each 50 µL reaction, add the master mix components followed by the DNA template.

Instructions:

1. Thaw all reagents on ice. Gently vortex and briefly centrifuge to ensure homogeneity.

2. Place thin-walled PCR tubes on ice. For each 50 µL reaction, add the master mix components followed by the DNA template.

3. Gently vortex each tube and briefly centrifuge to collect contents at the bottom.

4. If using a thermal cycler without a heated lid, overlay each reaction with 25 µL of mineral oil to prevent evaporation.

5. Run PCR using the cycling conditions described below.

Reactions Mix:

Reagent	Amount
Rai™ DNA polymerase (5.000 U/ml)	0.25 µl
UTaq Buffer	5 µl
dNTP Mix (10 mM)	1 µl
Forward Primer (10 µM)	2.5 µl*
Reverse Primer (10 µM)	2.5 µl*
25 mM MgCl2 (optional)	**
DNA template	1pg-1µg**
Water (Ultra Pure)	X***
Total Reaction Volume	50 µl

*Can be changed if needed for better results, recommended range is 0.1–0.5 µM final concentration

**Additional MgCl₂ can be added for better results. Note that UTaq buffer includes appropriate concentration of MgCl₂ for standard applications

***Ultra-pure water is added to bring the total reaction volume to 50 µl.

Thermal Cycling Conditions:

Step	Temperature	Time	Cycles
Initial Denaturation	95°C	1–3 min (up to 10 min for GC-rich DNA)	1
Denaturation	95°C	30 sec	25–40
Annealing	Tm – 5°C	30 sec
Extension	72°C	1 min/kb
Final Extension	72°C	5–15 min (up to 30 min for TA cloning)	1
Hold	4°C	∞

Component-Specific Recommendations

Template DNA

Plasmid/phage DNA: 0.01–1 ng per 50 µL reaction.

Genomic DNA: 0.1–1 µg per reaction.

Using excessive template may increase non-specific amplification, while too little may reduce yield. Avoid contaminants such as phenol, EDTA, or proteinase K which can inhibit polymerase activity. Ethanol precipitation and thorough 70% ethanol washes help remove these inhibitors.

Magnesium Chloride (MgCl₂)

Mg²⁺ is essential for polymerase activity and interacts with DNA, primers, and dNTPs. The optimal MgCl₂ range is 1–4 mM.

The UTaq buffer contains an appropriate concentration of MgCl₂ for optimal reaction.

If your DNA samples contain EDTA or other metal chelators, increase the Mg²⁺ concentration accordingly.

dNTPs

A final concentration of 0.2 mM for each dNTP is standard. Higher concentrations may be used for specific applications, but Mg²⁺ concentration must be adjusted due to its binding with dNTPs.

Primers

Use primers at 0.1–1 µM final concentration. Higher concentrations, especially with degenerate primers (0.3–1 µM), may help with efficiency, but excessive amounts can promote non-specific products.

Additional PCR Tips

Initial Denaturation: Required to fully denature template DNA, especially for GC-rich regions. Use 1–3 minutes at 95°C for normal templates or extend to 10 minutes for GC-rich DNA.

Denaturation Step: Standard 30 sec at 95°C is sufficient per cycle. For GC-rich DNA, increase to 3–4 minutes. Additives like DMSO (10%), formamide (5%), or betaine (1–1.5 M) can enhance denaturation but may require more Taq enzyme due to inhibition.

Annealing: Typically 5°C below primer Tm. Adjust in 1–2°C increments if non-specific bands appear. Lower temperatures may be needed if additives affecting Tm are used.

Extension: 1 minute per kilobase at 72°C. Extend longer for large amplicons.

Cycle Number: Use 25–35 cycles for standard reactions. Up to 40 cycles may be needed for low template inputs (<10 copies).

Final Extension: A 5–15 min step at 72°C ensures complete extension. Extend to 30 min if TA cloning is planned to promote 3′ A-overhangs.

No Amplification / No Product

•Check template DNA quality and quantity
•Redesign primers if Tm or specificity is problematic
•Increase MgCl₂ (try 2.0–3.0 mM)
•Extend elongation time (1 min per kb at 72°C)
•Lower annealing temperature (reduce by 2–5°C)
•Verify enzyme activity; avoid freeze-thaw; use fresh aliquot

Non-Specific Bands / Smearing

•Raise annealing temperature for higher specificity
•Reduce primer concentration (try 0.2–0.3 µM final)
•Avoid contamination—use fresh reagents and clean workspace
•Reduce cycle number (keep within 25–35 cycles)

Primer-Dimers

•Lower primer concentration
•Increase annealing temperature
•Redesign primers to eliminate 3′-complementarity
•Faint or Weak Bands
•Increase template input (up to 1 µg)
•Use high-quality DNA, free from inhibitors (e.g., ethanol, salts)
•Raise cycle number (up to 40 if needed)
•Double-check dNTPs and buffer amounts

 

For technical support, please contact us through;

[email protected]

Primer Design

Utilize primer design software or adhere to these general guidelines for PCR primer design: 

1. Aim for PCR primers that range from 15 to 30 nucleotides in length. 

2. The optimal GC content for the primer should be between 40% and 60%, with an even distribution of C and G nucleotides. 

3. Avoid having more than three consecutive G or C nucleotides at the 3’-end to minimize the chance of non-specific priming. 

4. Preferably, end the primer with a G or C at the 3’-end. 

5. Prevent hairpin formation and primer dimerization by avoiding self-complementary primer regions and complementarities between the primers, as well as direct primer repeats. 

6. Scrutinize potential sites of undesired complementarity between primers and template DNA. 

7. When crafting degenerate primers, ensure at least three conserved nucleotides are positioned at the 3’-end. 

8. Maintain a narrow range of melting temperatures (Tm) between the two primers, with differences not exceeding 5°C.

Below is a list of useful tools for primer analysis and melting temperature (Tm) calculation. Please note that these tools are provided by third-party companies that are not affiliated with Synbiotik Co. Synbiotik Co. does not accept any responsibility for the accuracy or outcomes of results generated by these tools;

Benchling
Geneious
IDT OligoAnalyzer
Eurofins Oligo Analysis Tool
NCBI Primer Design and Analysis Tool
PrimerDigital Web Tools for Primer Design and Analysis

 

For technical support, please contact us through [email protected]