How to Phusion PCR after Gibson assembly?

04 July 2023 1 1K Report

I am currently using NEBuilder Hifi DNA assembly to perform Gibson Assembly on several DNA fragments. The assembled fragment is okay, which is checked by gel electrophoresis. However, when I conduct PCR using Phusion enzyme on the assembled fragment, only one band that is much shorter than the expected band appears. Could anyone explain why and how I can solve this problem?

Tlehema Umbayda

Phusion PCR is a DNA amplification technique commonly used in molecular biology. you can follow these general steps:

Design primers

Set up the PCR reaction: make sure you have the following: Phusion DNA polymerase, dNTPs (deoxynucleotide triphosphates), PCR buffer, template DNA (Gibson assembly product), and forward and reverse primers.

PCR cycling conditions: Set up the PCR cycling conditions for your Phusion PCR reaction.

PCR product analysis: Analyze the PCR products by running an aliquot of the reaction mixture on an agarose gel or using other appropriate methods such as capillary electrophoresis.

Purification of PCR product: Purify the PCR product using a suitable method to remove primers, unincorporated nucleotides, and other PCR components. Options include commercial PCR purification kits, gel extraction kits, or enzymatic clean-up methods.

Optional verification: If necessary, you can confirm the correctness of the PCR product by sequencing.

Remember to optimize the PCR conditions, such as primer concentrations, annealing temperatures, and extension times, if necessary, to obtain the best results. If necessary, remember to optimize the PCR conditions, such as primer concentrations, annealing temperatures, and extension times

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