TEMPase Hot Start Master Mix BLUE

PCR Hot start PCR - Bacterial DNA

Experiment
PCR Hot start PCR - Bacterial DNA
Product
TEMPase Hot Start Master Mix BLUE from Ampliqon
Manufacturer
Ampliqon

Protocol tips

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Working on ice is not required.

Publication protocol

Genomic DNA was extracted using the Dynabeads DNA DIRECT Universal kit (Invitrogen) on a 24 h culture. For each PCR reaction, 1 μL of extracted genomic DNA was used with primers listed in . PCR for the species identification and MLST were performed using the TEMPase Hot Start 2X Master Mix Blue II (Ampliqon, Denmark) and the Maxima Hot Start Taq Polymerase (Fermentas, Waltham, MA, United States) was used for toxin-encoding gene amplification, both were performed in a 25 μL reaction following the manufacturer’s instruction. Reactions were done in a Veriti Thermal Cycler (Applied Biosystems, 96 Well Model 9902). PCR products were purified using 10 U Exonuclease I (Thermo Fisher Scientific, Waltham, MA, United States) and 0.5 U Fast Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, United States) and incubated at 37°C for 15 min followed by an inactivation step at 85°C for 15 min (). Macrogen Europe (The Netherlands) sequenced the gene fragments using the same primer used for the PCR amplification.

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Reviews

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Discussion

Discussion

5 years ago

Author: Qatar

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

Discussion

5 years ago

Author: Janina Reinhardt Switzerland

Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Hot start PCR - Bacterial DNA using TEMPase Hot Start Master Mix BLUE from Ampliqon.

Paper title
in Some Brazilian Dairy Industries: Changes of Contamination and Diversity
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Manufacturer protocol

Download the product protocol from Ampliqon for TEMPase Hot Start Master Mix BLUE below.

Download PDF Download manufacturer protocol

Videos

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