EzPCR™ GC 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, orange G, and stabilizer). PCR can be performed simply by adding primer pair and template. EzPCR™ GC 5x PCR Master Mix is designed for high-fidelity PCR amplification of GC-rich templates (>70% or greater GC content) and is based on high-fidelity HiPi Plus DNA polymerase.

   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 complex targets with a robust yield and high specificity. HiPi plus DNA polymerase is recommended for use in conventional PCR that products are less than 5 kb (for lambda DNA).  HiPi Plus DNA Polymerase generates a mixture of PCR products with blunt end and 3’ -dA overhangs.

  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).

 

Applications

 - Designed to perform PCR amplification on all DNA templates.

 - Amplification of cDNA and genomic DNA

 - High GC content PCR 

 

Qualifying Test

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

 

Storage Condition

  Store at -20 ℃.

  EzPCR™ GC 5x PCR Master Mix is stable for at least 1 years 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™  GC 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 Kbp
Denature	95 ℃	10 - 30 sec	20 - 30 sec
Anneal	Tm - 4 ℃	10 - 20 sec	20 - 30 sec
Extend	72 ℃	10 - 30 sec	30 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 orange G (rose gold 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 pg - 10 ng of plasmid DNA and 10-100 ng of genomic DNA are 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.