Cloning and Sequencing of Lignocellulose-Degrading Enzymes

Using cDNA obtained by reverse transcription as a template, RT-PCR amplification of xynF1 (NCBI ID: 4,980,082), abnC (NCBI ID: 4,979,546), cbhC (NCBI ID: 4,982,491), bglM (NCBI ID: 4,984,238), and eglD (NCBI ID: 4,988,091) was carried out using gene-specific primers. The PCR products were purified using the DNA Gel Extraction Kit (Sangon Biotech Co., Ltd., Shanghai, China), ligated into the One Step ZTOPO-Blunt/TA vector (Zhuangmeng Technology Co., Ltd., Beijing, China) and transformed into Escherichia coli DH5α. The clones were verified by DNA sequencing (Sangon Biotech Co., Ltd., Shanghai, China). Then, intron-spanning primers were designed for PCR validation based on the five lignocellulose-degrading enzyme genes xynF1, abnC, cbhC, bglM and eglD. The primers are listed in the Supplemental Table S1.

Free full text: Click here

Xu Y., Dong F., Wang R., Ajmal M., Liu X., Lin H, & Chen H. (2024). Alternative splicing analysis of lignocellulose-degrading enzyme genes and enzyme variants in Aspergillus niger. Applied Microbiology and Biotechnology, 108(1), 302.

Publication 2024

DNA Gel Extraction Kit DNA sequencing

Corresponding Organization :

Other organizations : Henan Agricultural University

Top 5 similar protocols

Variable analysis

independent variables

Reverse transcription
RT-PCR amplification of xynF1, abnC, cbhC, bglM, and eglD genes

dependent variables

Expression levels of xynF1, abnC, cbhC, bglM, and eglD genes

control variables

CDNA obtained by reverse transcription as a template
Gene-specific primers used for RT-PCR amplification
DNA Gel Extraction Kit for PCR product purification
One Step ZTOPO-Blunt/TA vector for ligation and transformation into Escherichia coli DH5α
DNA sequencing for clone verification
Intron-spanning primers for PCR validation

positive controls

None specified

negative controls

None specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!