Chromosome Translocation Detection by Southern Blot

DNA from CHEF gels were transferred overnight to a Zeta-Probe GT Membrane (Bio-Rad, catalog no. 162-0196) in 20× SSC and cross-linked using UV (150 mJ). Probing and detection of the DNA were conducted as previously described (45 (link)). Briefly, probes were generated by PCR incorporation of DIG-11-dUTP into target sequences according to the manufacturer’s instructions (Roche). Chromosome 5-to-chromosome 7 translocation was detected using a probe generated with the primers AB1028 and AB1029. The probe hybridized to two different chromosomes: in strain Y-11545, the probe hybridized with Chr5 (nt 448855 to 449034), and in strain Y-7124, the probe hybridized with Chr7 (nt 494698 to 494877) (see Table S2). The membrane was incubated with the probe overnight at 42°C with DIG Easy Hyb (Roche, catalog no. 11603558001). The DNA was detected with anti-digoxigenin-alkaline phosphatase antibody (Roche, catalog no. 11093274910) and CDP Star Ready-to-Use (Roche, catalog no. 12041677001) according to the manufacturers’ instructions.

Free full text: Click here

Vega-Estévez S., Armitage A., Bates H.J., Harrison R.J, & Buscaino A. (2021). The Genome of the CTG(Ser1) Yeast Scheffersomyces stipitis Is Plastic. mBio, 12(5), e01871-21.

Publication 2021

Zeta-Probe GT Membrane DIG Easy Hyb CDP Star Ready-to-Use

Alkaline phosphatase Anti antibody Chromosome translocation Chromosomes Digoxigenin Dutp Gels Membrane Primers Strain

Corresponding Organization : University of Kent

Other organizations : University of Greenwich, Natural Resources Institute, National Institute of Agricultural Botany

Top 5 similar protocols

Variable analysis

independent variables

DNA transfer from CHEF gels to Zeta-Probe GT Membrane
UV cross-linking of DNA (150 mJ)
Probe generation by PCR incorporation of DIG-11-dUTP into target sequences
Probe used to detect chromosome 5-to-chromosome 7 translocation

dependent variables

Chromosome 5-to-chromosome 7 translocation detection

control variables

Probe hybridization conditions (42°C, DIG Easy Hyb)
Detection method (anti-digoxigenin-alkaline phosphatase antibody, CDP Star Ready-to-Use)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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!