EzPCR™ LF 5X 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 5X concentration format, users should adjust a final reaction to 1X (if final reaction volume is 20 ㎕, 4 ㎕ of 5X Master Mix should be added).

  One unit of HiPi Plus DNA polymerase is contained in 4 ㎕ of 5X PCR Master Mix. HiPi Plus DNA polymerase in Master Mix is suitable for a high fidelity amplification of DNA fragments.

This enzyme is designed for a reliable amplification of long (up to 10 Kbp for lambda DNA) and complex targets with a robust yield and high specificity. HiPi Plus DNA Polymerase generates a mixture of PCR products with blunt end and 3’ -dA overhangs. 

 

Applications

 - Long and Fast PCR

 - RT-PCR

 - Amplification of cDNA and genomic DNA

 - High fidelity PCR for a cloning 

 

Qualifying Test

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

 

Storage Condition

  Store at -20 ℃.

  EzPCR™ LF 5X PCR Master Mix is stable for at least 1years at recommended storage condition. 

 

Features

 - 5’ → 3’ exonuclease : no

 - 3’ → 5’ exonuclease : weak

 - strand displacement : no 

 

Standard Experiment Protocol

1. Prepare the 20 ㎕ PCR solution as follows

EzPCR™ LF 5X Master Mix                         4 ㎕ 

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 ℃	20 sec	20 sec
Anneal	Tm - 4 ℃	10 - 20 sec	10 - 20 sec
Extend	72 ℃	20 sec	20 - 40 sec / Kbp

* Set 25-35 PCR cycles for effective amplification.

 

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.