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Cat. No.: YCPKEL-100 (for 100T)

Cat. No.: YCPKEL-500 (for 500T)

Description

Yeast Colony PCR Kit (Enzymatic Lysis) is a highly convenient kit used for PCR screening and identification of positive clone colonies of yeast strains such as brewing yeast and Saccharomyces cerevisiae after plasmid transformation.

This kit provides Yeast Lysis Enzyme for digesting yeast cell walls, and Yeast Colony PCR Mix (Green, 2X) for PCR amplification. Yeast Colony PCR Mix (Green, 2X) is a PCR premix with a double concentration of blue and orange dyes (approximately at positions 4kb and 50bp in electrophoresis), containing 2X Taq DNA Polymerase, 2X PCR Buffer, 2X dNTPs, and 2X Loading Buffer. Users only need to add yeast colony lysate, primers, and water for PCR amplification, and after amplification, samples can be directly loaded onto agarose gel for electrophoresis.

Yeast, a classic fungal model organism, is widely used in studies of cell biology, genetics, aging, and more. As a eukaryotic organism, yeast possesses a fast growth rate, mature post-translational modification systems, and resistance screening systems. It is often used as a heterologous host detection system for protein expression and interaction in molecular biology research involving animals and plants. When performing genetic manipulations in yeast, it is necessary to identify the introduction or deletion of DNA fragments to ensure accuracy. Conventional steps include single colony amplification culture, cell collection and lysis, extraction of high-purity DNA templates, and PCR detection. However, this method is often costly and inefficient for large-scale screening. Colony PCR eliminates the need for prior separation or purification of DNA, directly amplifying the target sequence from yeast cells or lysates, making it relatively rapid and efficient.

The cell wall of yeast is mainly composed of polysaccharides (85%-90%) and proteins (10%-15%), with polysaccharides consisting of mannoproteins, β-glucans, and small amounts of chitin. β-glucans in the inner layer of the cell wall are covalently linked to chitin, forming the cell scaffold. Mannoproteins in the outer layer are linked to mannans in the middle layer via O-glycosidic bonds, forming a mannoprotein layer covering the cell surface, providing yeast cells with toughness. The unique structure of the yeast cell wall makes it difficult for DNA to be released simply by PCR heating, resulting in low success rates for many PCR kits that directly use yeast as a template.

This kit provides Yeast Lysis Enzyme for effective pretreatment of yeast before PCR, allowing for complete degradation of yeast cell walls and release of yeast DNA, ensuring effective yeast colony PCR, thereby significantly reducing the time required for identification of positive clones after yeast transformation and improving experimental efficiency.

Components

• Yeast Lysis Enzyme
• Enzyme Preparation Solution
• Enzyme Reaction Buffer
• Yeast Colony PCR Mix (Green, 2X)

Features

• This kit is extremely convenient to use. The Yeast Colony PCR Mix (Green, 2X) provided in the kit already contains all the common components. Users only need to prepare primers and water to perform PCR amplification on yeast colonies. It greatly simplifies PCR operations, making them faster and reducing the potential for contamination during PCR, thus improving the repeatability of PCR. Additionally, the Yeast Colony PCR Mix (Green, 2X) already includes loading buffer components. After PCR amplification, samples can be directly loaded onto agarose gel for electrophoresis without the need to add loading buffer.
• The kit provides dual-color dyes, facilitating observation of the electrophoresis process. The Yeast Colony PCR Mix (Green, 2X) in the kit contains blue and orange electrophoresis tracking dyes (appearing green overall), with migration positions at approximately 4kb and 50bp in a 1% agarose gel, respectively. After PCR, direct electrophoresis can be performed without the need to add loading buffer. The blue and orange tracking dyes do not interfere with the observation and detection of corresponding DNA bands.
• The product exhibits high stability. Testing has shown that the PCR mix in this kit remains effective for PCR amplification after undergoing 15 freeze-thaw cycles with no significant impact.

Storage

Store at -20°C, effective for at least one year. Enzyme Preparation Solution and Enzyme Reaction Buffer can be stored at room temperature or 4°C. Once prepared into a solution, Yeast Lysis Enzyme can be stored at 4°C for one month or at -20°C for six months.

Precaution

• Yeast colonies are difficult to lyse. When lysing, please ensure thorough mixing by pipetting or vortexing.
• Since PCR reactions are highly sensitive and can amplify the target gene over 10 million times, please ensure to avoid contamination of trace amounts of DNA when setting up PCR reactions. Consider setting up blank controls without template DNA to confirm the absence of DNA contamination.
• Although the PCR mix in this product retains nearly identical PCR amplification efficiency after 15 freeze-thaw cycles, it is advisable to avoid repeated freeze-thaw cycles as much as possible, as multiple cycles may degrade product performance.
• Before using this product, ensure complete thawing and gently invert the tube to mix thoroughly, while minimizing the formation of bubbles.
• This product is intended for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food, or drug purposes. It should not be stored in residential settings.
• For your safety and health, wear laboratory coats and disposable gloves when handling.

Related:

• Yeast Colony PCR Kit (Alkaline Lysis)
• Yeast Colony PCR Kit (Enzymatic Lysis)
• Competent Cell Preparation and Transformation Kit for Pichia Pastoris
• Competent Cell Preparation and Transformation Kit for Saccharomyces Cerevisiae

Only for research and not intended for treatment of humans or animals

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