Rapid PCR amplification kit for yeast colonies and method for performing PCR amplification on yeast colonies by using kit

Abstract

The invention discloses a rapid PCR amplification kit for yeast colonies and a method for performing PCR amplification on the yeast colonies by using the kit. The kit comprises: 10 x cell lysis buffer, 10 x PCR reaction solution, dNTP and hot-start Taq enzyme. Compared with the prior art, the kit does not need the genome extraction steps of heating, boiling, phenol / chloroform extraction, and the like, so that the toxicity of toxic substances to experimental personnel is reduced. The method has quick and simple operation and saves a great deal of time. The method directly lyses monoclonal colonies, reduces cross pollution, and avoids false positivity. By using the cell wall lysis buffer with high pH value, the cell wall of the yeast can be effectively lysed in a short time, the yeast genomecan be fully released, the pH value of the released supernatant after being mixed with the matched PCR reaction solution is within a proper range of PCR amplification, and the rapid PCR amplificationcan be realized.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a yeast colony rapid PCR amplification kit and a method for using the kit to PCR amplify yeast colonies. Background technique [0002] Yeast is a kind of fungal microorganism, which is widely distributed in nature and is a typical facultative anaerobic microorganism. It can ferment sugar into alcohol and carbon dioxide ponds, and is widely used in the food field. Secondly, as an important engineering bacterium, It is widely used in the molecular field, for example in the field of molecular cloning as eukaryotic recipient cells. In yeast molecular cloning, PCR is required to verify whether the exogenous gene has been successfully transformed after the transformation of the exogenous gene into the yeast. When performing colony PCR with traditional bacteria, you can directly pick a single bacterial colony or a bacterial solution, but yeast is different from bacte...

AI Technical Summary

Problems solved by technology

When performing colony PCR with traditional bacteria, you can directly pick a single bacterial colony or a bacterial solution, but yeast is different from bacteria, and its cell wall is difficult to break. Improper treatment and insufficient genome release will cause PCR amplification failure.

[0003] When performing PCR amplification on the yeast gene, the traditional method is to extract the total genome of the yeast and then perform PCR amplification. This method can obtain a relatively high-purity genome for PCR amplification and the amplification effect is ideal. However, for the conversion rate of yeast, researchers may need to screen and identify more colonies. Extracting the genome will not only take a lot of time and increase the cost of the experiment, but also in the process of genome extraction, due to the large number of samples, the operation is slightly inconvenient. If you are careful, it will cause cross-contamination between samples, which will bring troubles to subsequent experiments.

[0004] At present, yeast colony PCR technology is widely used, which eliminates the cumbersome steps of genome extraction, mainly pre-processing the cell wall of yeast, including repeated freezing and thawing to break the wall, enzyme digestion treatment, high-temperature cooking, and extended PCR. Pre-denaturation time, etc. Although the yeast treated by these methods can effectively perform PCR amplification, the pretreatment time is relatively long, the operation is cumbersome, and the stability and amplification need to be further improved.

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