EzPCR™ FAST 2X PCR Master Mix is a ready-to-use premix containing all the components essential for a PCR and agarose gel electrophoresis (DNA polymerase, dNTP, reaction buffer, glycerol, bromophenol blue, and stabilizer). PCR can be performed simply by adding primer pair and template.

  As Master Mix is supplied as a 2X concentration format, users should adjust a final reaction to 1X (if final reaction volume is 20 ml, 4 ml of 2X Master Mix should be added). One unit of rTaq Plus DNA Polymerase is contained in 4 ml of 2X Master Mix.

  rTaq Plus DNA Polymerase is a thermostable DNA polymerase that designed for a reliable amplification of long and complex targets with a robust yield in an extremely short extension time. Due to its high elongation rate (about 100 bp/sec) and high processivity (about 250 bases), long sized products can be generated within relatively shorter running time compared to any other commercially available enzymes (5 kbp within 60 sec extension).

  rTaq Plus DNA Polymerase has no detectable exonuclease activity (5´à3´ or 3´à5´) and generates a 3´dA overhang, suitable for a TA cloning of PCR products.

 

Applications

 - Fast PCR

 - RT-PCR

 - Amplification of cDNA and genomic DNA

 - Colony PCR

 

Qualifying Test

   Performance test, exo/endo nuclease contamination test, stability test. genomic DNA contamination test. 

 

Storage Condition 

   Store at -20 ℃.

   EzPCRTM FAST 5X PCR Master Mix is stable for at least 1years at recommended storage condition. 

 

Features

- 5’ → 3’ exonuclease : no

- 3’ → 5’ exonuclease : no

- strand displacement : no

 

Standard Experiment Protocol

1. Prepare the 20 ㎕ PCR solution as follows

EzPCR™ FAST 5X Master Mix                      10 ㎕ 

Primer (10 pmol/㎕)                           each 0.5 ㎕

Template DNA                                      0.1 - 10 ng
 PCR grade distilled water                             - ㎕

  

Add distilled water to make 20 ㎕ final volume.

 

2. Set PCR cycling as follows

Initial denature at 95 ℃ : 3 min

		< 1 Kbp	1 - 10 Kbp
Denature	95 ℃	5 sec	10 sec
Anneal	Tm - 4 ℃	5 sec	10 sec
Extend	72 ℃	5 sec	20 sec / Kbp

* Set 25-35 PCR cycles for effective amplification.

* You can also use two step cycle amplification. (denaturation at 95℃ andannealing / extension at 68℃) 

 

3. You can analyze PCR products by direct loading into agarose gel because PCR Master Mix contains   glycerol and bromophenol blue (blue color) essential for a gel loading.

 

Troubleshooting

 

1. No products

 - Confirmyourtemplateisintact:Try another reaction with a result assured primer pair and templates.

 

2. Smear bands or smeared background

 - Reduce template concentration: High concentration of template can lead to smearing of PCR products. Generally, 1 - 10 ng of plasmid DNA  Is working well

 - Increase annealing temperature

 - Set up a reaction mix on ice

 

3. Non-specific bands

 - Increase annealing temperature

 - Consider using PCR additives, like 1-2% DMSO or 0.5 - 1 X Q Buffer

 - Confirm specificity of your primers

 - Adjust concentration of template

 

4. Low yield

 - Increase PCR cycle number

 - Be sure appropriate concentration of your template is added.