EzPCR™ XO 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, phenol red and stabilizer). PCR can be performed simply by adding primer pair and template.
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 2x concentration format, users should adjust a final reaction to 1x (if final reaction volume is 20 ㎕, 10 ㎕ of 2x Master Mix should be added).
Applications
- Long 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™ XO 2x 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™ XO 2x 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
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< 1 Kbp
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> 1 Kbp
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Denature
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95 ℃
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10 - 30 sec
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20 - 30 sec
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Anneal
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Tm - 4 ℃
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10 - 20 sec
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20 - 30 sec
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Extend
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72 ℃
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10 - 30 sec
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30 sec / Kbp
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* 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 phenol red (red color) essential for a gel loading.
Troubleshooting
1. No products
- Confirm your template is intact : 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.
