USER GUIDE: EBL PCR Master Mix (2X), Red dye
Storage at 4°C for 6 months and -20°C for long term.
Description:
EBL Master Mix contain all components for PCR, only template DNA and primers should be added, volume adjusted by water if necessary and PCR can be started. 2X EBL PCR Mix is optimized mixture contain of Taq enzyme, reaction buffer, dNTPs and enhancer as 2-fold concentration. The PCR Master Mix is designed to allow the user for quick and easy preparation of reaction mixture. The PCR Master Mix may be amplification PCR products to 5 kb and the products can be directly cloning into T-vector.
Composition of the 2x PCR Master Mix
150 mM Tris-HCl pH 8.5, 40 mM (NH4)2S04, 3 mM MgCl2, and 0.2% Tween 20
0.4 mM of each dNTPs, Taq DNA polymerase Inert red dye and a stabilizer
Standard Protocol
This protocol serves as a guideline for primer extensions. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be determined individually.
1. Set up each reaction as follows:
|
Component Final
|
Vol./reaction*
|
Concentration*
|
|
2x PCR Master Mix
|
25 µl
|
1x
|
|
Primer F
|
X µl
|
0.2 µM (0.1 – 1.0 µM)
|
|
Primer R
|
X µl
|
0.2 µM (0.1 – 1.0 µM)
|
|
Water
|
X µl
|
|
|
DNA
|
X µl
|
genomic DNA: 50 ng (10 – 500 ng) plasmid DNA: 0.5 ng (0.1 – 1 ng) bacterial DNA: 5 ng (1 – 10 ng)
|
|
Total volume
|
50 µl
|
|
2. Mix gently by pipetting the solution up and down a few times.
3. Program the thermal cycler according to the manufacturer’s instructions. See table 2 for an example. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.4. Place the tubes in the thermal cycler and start the reaction.
Table 2. Three-step PCR program
|
Cycles
|
Duration of cycle
|
Temperature
|
|
1
|
2 – 5 minutes
|
95 °C
|
|
25-35
|
20 – 30 seconds
|
95 °C
|
|
|
20 – 40 seconds
|
50 – 65 °C
|
|
|
60 seconds/ 1Kb
|
72 °C
|
|
1
|
5 minutes
|
72 °C
|
Set up reaction mixtures in an area separate from that used for DNA preparation or product analysis. Work on ice at all times.
The final MgCl2 concentration of this 2x Taq Master Mix RED is 1.5 mM. In some applications, more than 1.5 mM MgCl2 is required for best results.
MCT-02125
USER GUIDE: EBL HP PCR Master Mix (2X), Red dye
Storage at 4°C for 6 months and -20°C for long term.
Size: 1.25ml
EBL HP PCR Master Mix contain all components for PCR, only template DNA and primers should be added, volume adjusted by water if necessary and PCR can be started. 2X EBL HP PCR Master Mix is optimized mixture contain of Taq enzyme, reaction buffer, dNTPs and enhancer as 2-fold concentration. EBL HP PCR Master Mix offers several advantages: High performance thermostable DNA polymerase, Increased reproducibility, Quick and easy preparation of reaction mixture and Direct loading PCR products into agarose gels. The PCR Mix may be amplification PCR products to 5 kb and the products can be directly cloning into T-vector.
Composition of the 2x PCR Master Mix
150 mM Tris-HCl pH 8.5, 40 mM (NH4)2S04, 3 mM MgCl2, and 0.2% Tween 20
0.4 mM of each dNTPs, Taq DNA polymerase Inert red dye and a stabilizer
Standard Protocol
This protocol serves as a guideline for primer extensions. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be determined individually.
1. Set up each reaction as follows:
|
Component Final
|
Vol./reaction*
|
Concentration*
|
|
2x PCR Master Mix
|
25 µl
|
1x
|
|
Primer F
|
X µl
|
0.2 µM (0.1 – 1.0 µM)
|
|
Primer R
|
X µl
|
0.2 µM (0.1 – 1.0 µM)
|
|
Water
|
X µl
|
|
|
DNA
|
X µl
|
genomic DNA: 50 ng (10 – 500 ng) plasmid DNA: 0.5 ng (0.1 – 1 ng) bacterial DNA: 5 ng (1 – 10 ng)
|
|
Total volume
|
50 µl
|
|
3. Program the thermal cycler according to the manufacturer’s instructions. See table 2 for an example. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.
4. Place the tubes in the thermal cycler and start the reaction.
Table 2. Three-step PCR program
|
Cycles
|
Duration of cycle
|
Temperature
|
|
1
|
2 – 5 minutes
|
95 °C
|
|
25-35
|
20 – 30 seconds
|
95 °C
|
|
|
20 – 40 seconds
|
50 – 65 °C
|
|
|
60 seconds/ 1Kb
|
72 °C
|
|
1
|
5 minutes
|
72 °C
|
Notes
Set up reaction mixtures in an area separate from that used for DNA preparation or product analysis. Work on ice at all times.
The final MgCl2 concentration of this 2x Taq Master Mix RED is 1.5 mM. In some applications, more than 1.5 mM MgCl2 is required for best results.