The total genomic DNA of Z. mays (inbred line Mo17) and Z. perennis (CIMMYT accession No. 9475) were isolated from young leaves using a modified 2× CTAB method (Fu et al. 2015 ), and then labeled with probes with a DIG-High Prime Kit and a Biotin Nick Translation Kit (Roche, Basel, Switzerland), respectively, according to the manufacturer’s instructions. The McGISH technique was performed according to the methods of Iqbal et al. (2019) (link). The McGISH images were visualized via a phase video microscope (Olympus BX61, Tokyo, Japan) equipped with a charge-coupled-device camera (700 mm CCD) and via Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA).
Biotin nick translation kit
Manufactured by Roche
Sourced in Switzerland, Germany
The Biotin nick translation kit is a laboratory tool designed for the incorporation of biotin-labeled nucleotides into DNA strands. This kit provides the necessary reagents and enzymes to perform the nick translation process, which is a technique used for the labeling of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Dig nick translation kit, by Roche (2 mentions)
Digoxigenin, by Roche (2 mentions)
Dig high prime kit, by Roche (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Lcs sp2, by Leica camera (1 mentions)
Cw4000, by Leica camera (1 mentions)
Axiovision ver 4, by Zeiss (1 mentions)
Ribonuclease a, by Qiagen (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axiovision version 4, by Zeiss (1 mentions)
Axio imager m2 epifluorescent microscope, by Zeiss (1 mentions)
8 protocols using biotin nick translation kit
1
Maize Genomic DNA Isolation and McGISH
2
Quantifying Satellite DNA Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dot-blot hybridization was used to estimate the amount of the Hyscu-H satDNA in the H. scutellatus genome [59 (link),60 (link)]. Inserts of the recombinant Hyscu-H-20 and Hyscu-H-21 plasmids were labeled with DIG as indicated above and used as a probe in the dot-blot hybridization.
Physical location of the rDNA clusters was determined by DNA FISH. The plasmid pDmra.51#1, with a noninterrupted 11.5 kb rDNA unit containing 18S and 28S genes of Drosophila melanogaster [61 (link)], was used as a probe. For location of the Hyscu-H satDNA family repeats, the inserts of the recombinant plasmids were used as a probe. Probes were labeled with biotin-16-dUTP using the biotin nick translation kit (Roche Diagnostics GmbH, Mannheim, Germany). FISH was carried out following the procedure described by Palomeque et al. [62 (link)] using the biotin-labeled probe (2 ng probe/mL, 50% formamide). FISH probe detection was performed using the avidin-FITC/anti-avidin-biotin system with two amplification rounds for the satDNA probe and three rounds for the rDNA probe. Slides were mounted using VECTASHIELD® with DAPI (Vector Labs, Burlingame, CA, USA).
Physical location of the rDNA clusters was determined by DNA FISH. The plasmid pDmra.51#1, with a noninterrupted 11.5 kb rDNA unit containing 18S and 28S genes of Drosophila melanogaster [61 (link)], was used as a probe. For location of the Hyscu-H satDNA family repeats, the inserts of the recombinant plasmids were used as a probe. Probes were labeled with biotin-16-dUTP using the biotin nick translation kit (Roche Diagnostics GmbH, Mannheim, Germany). FISH was carried out following the procedure described by Palomeque et al. [62 (link)] using the biotin-labeled probe (2 ng probe/mL, 50% formamide). FISH probe detection was performed using the avidin-FITC/anti-avidin-biotin system with two amplification rounds for the satDNA probe and three rounds for the rDNA probe. Slides were mounted using VECTASHIELD® with DAPI (Vector Labs, Burlingame, CA, USA).
3
Chromosome Identification in Hybrid Fish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two probes were used for fluorescence in situ hybridization (FISH): a species-specific centromere probe that was designed using the RCC genome and amplified by PCR using the primers 5′-TTCGAAAAGAGAGAATAATCTA-3′ and 5′-AACTCGTCTAAACCC GAACTA-3′, and a 5S gene probe that was constructed using the RCC genome and amplified by PCR using the primers 5′-GCTATGCCCGATCTCGTCTGA-3′ and 5′-CAGGTTGGTATGGCCGTAAGC-3′ (Qin et al. 2010 (link)). The species-specific centromere probes were labeled with biotin-16-dUTP (using a biotin-nick translation kit, Roche, Germany), and the 5S gene probes were labeled with DIG-11-dUTP (using a DIG-nick translation kit, Roche, Germany). FISH was performed according to He et al. (2012 (link)). Two hundred metaphase chromosome spreads from ten individuals were analyzed for each type of fish (RCC, BSB, 4nRB, 4nRR, 2nG, and 2nH). Preparations were examined under an inverted microscope (CW4000, Leica, Germany) with a confocal imaging system (LCS SP2, Leica). Captured images were colored and superimposed in Adobe Photoshop CS6.
4
Genomic In Situ Hybridization of Allopolyploid Bananas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GISH has been performed in one diploid hybrid individual B6B7 (H258) and four allopolyploid individuals, two of AAB7B7 (H110–1 & 2) and two of B6B6B7B7 (H574–1 & 2) composition, using labelled parental diploid genomic DNA as probes [24 (link),29 (link)]. Total genomic DNA from diploid cytotypes AA, B6B6, and B7B7 was isolated using the CTAB (Cetyltrimethylammonium Bromide) method [29 (link)] and sheared at 98 °C for 5 min. Approximately 1 μg of genomic DNA of each cytotype was labeled using either digoxigenin or a biotin nick translation kit (Roche). GISH was carried out following the method of [29 (link)].
All preparations after FISH and GISH were analysed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), and images were captured with a CCD camera and processed using AxioVision ver. 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.
All preparations after FISH and GISH were analysed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), and images were captured with a CCD camera and processed using AxioVision ver. 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.
5
FISH Probe Labeling and RNA Exclusion
6
Chromosome Spread Preparation and FISH Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromosome spreads were obtained from adult male gonads following the method described by Lorite et al. [23 (link)]. The DNA probe for FISH was generated by a polymerase chain reaction (PCR) as described previously by Lorite et al. [24 (link)], using (TTAAAA)4 and (AATTTT)6 oligonucleotides as primers without template. These primers, when mixed, annealed to two stable double-stranded DNA forms with 3´and 5´ protruding ends. PCR generated fragments between 200 and 1000 bp fragments that were labelled with biotin-16-dUTP using the biotin nick translation kit (Roche Diagnostics GmbH, Mannheim, Germany). FISH was carried out following the procedure described by Lorite et al. [24 (link)] and Palomeque et al. [25 (link)] using the biotin labelled probe (2 ng probe/mL, 50% formamide). Fluorescence immunological detection was performed using the avidin-FITC/anti-avidin-biotin system without amplifications rounds. Slides were counterstained with propidium iodide and 4′-6-diamino-2-fenil-indol (DAPI).
7
Genomic in situ Hybridization of Melampodium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Actively growing root meristems were harvested, pretreated with 0.002 M solution of 8-hydroxyquinoline for 2.5 h at room temperature and 2.5 h at 4 C, fixed in a 3:1 ethanol and acetic acid mixture, and stored at 20 C until use (Weiss-Schneeweiss et al. 2012 (link)). Chromosome preparations were made after enzymatic digestion of fixed root meristems as described earlier (Weiss-Schneeweiss et al. 2012 (link)).
Genomic in situ hybridization was performed for the allotetraploid M. strigosum (M147, Hidalgo, Mexico) and for the allohexaploids M. sericeum (M63, Oaxaca, Mexico) and M. pringlei (M2089, Oaxaca, Mexico), using gDNAs of previously identified parental taxa (Weiss-Schneeweiss et al. 2012 (link)) as probes. Parental gDNAs of diploid Melampodium linearilobum, M. glabribracteatum, and M. americanum, as well as allotetraploid M. strigosum were sheared at 98 C for 5 min and labeled using either digoxigenin or the biotin nick translation kit (Roche, Vienna, Austria). The recently developed formamide-free hybridization and detection technique (Jang and Weiss-Schneeweiss 2015 (link)) was applied for GISH. Preparations were analyzed with an Axiolmager M2 epifluorescent microscope (Carl Zeiss). Images were captured with a CCD camera and processed using AxioVision 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.
Genomic in situ hybridization was performed for the allotetraploid M. strigosum (M147, Hidalgo, Mexico) and for the allohexaploids M. sericeum (M63, Oaxaca, Mexico) and M. pringlei (M2089, Oaxaca, Mexico), using gDNAs of previously identified parental taxa (Weiss-Schneeweiss et al. 2012 (link)) as probes. Parental gDNAs of diploid Melampodium linearilobum, M. glabribracteatum, and M. americanum, as well as allotetraploid M. strigosum were sheared at 98 C for 5 min and labeled using either digoxigenin or the biotin nick translation kit (Roche, Vienna, Austria). The recently developed formamide-free hybridization and detection technique (Jang and Weiss-Schneeweiss 2015 (link)) was applied for GISH. Preparations were analyzed with an Axiolmager M2 epifluorescent microscope (Carl Zeiss). Images were captured with a CCD camera and processed using AxioVision 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.
8
Genomic In Situ Hybridization of Nicotiana
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cytological investigations, actively growing root meristems of Nicotiana sect. Repandae species were pre-treated with 0.002 M solution of 8-hydroxyquinoline for 2.5 h at room temperature and 2.5 h at 4 °C, fixed in ethanol-to-acetic acid (3:1) and stored at −20 °C until use. Genomic in situ hybridisation was performed using genomic DNAs of previously identified closest extant relatives of parental diploid species (N. sylvestris and N. obtusifolia; both 2n = 24) as probes (Clarkson et al. 2005 (link); Lim et al. 2007 (link); Renny-Byfield et al. 2013 (link)). Genomic DNAs of diploid species were isolated using the CTAB method (Jang and Weiss-Schneeweiss 2015 (link)), sheared at 98 °C for 5 min and labelled using either digoxigenin or biotin nick translation kit (Roche). Hybridisation and detection of the probes followed the method of Jang and Weiss-Schneeweiss (2015 (link)). Preparations were analysed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), images acquired with a CCD camera and processed using AxioVision version 4.8 (Carl Zeiss, Vienna, Austria) with only those functions that apply equally to whole image. At least 30 well-spread metaphases and prometaphases were analysed for each individual. Cut-out karyotypes are given in Online Resource 1.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!