Microdissection of sex chromosomes was performed according to described protocol [27 (link)] with some modifications. For each DNA probe, 15–20 copies of the chromosome or chromosome region were collected by extended glass needle using a micromanipulator MR (Zeiss, Oberkochen, Germany). Proteinase K treatment and low temperature cycles of degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) with Sequenase 2.0 (ThermoFisher Scientific, Waltham, MA, USA) were performed exactly as described [27 (link)]. High temperature cycles of DNA amplification were carried out with Encyclo Polymerase mix (Evrogen, Moscow, Russia) in 33 cycles of PCR according to the producer’s recommendation. DNA probes were labeled in an additional 25 cycles of PCR with Tamra-5-dUTP and Fluorescein-12-dUTP (Biosan, Novosibirsk, Russia) as described [28 (link)].
Tamra 5 dutp
Manufactured by Biosan
Tamra-5-dUTP is a nucleotide analog used in fluorescence-based applications. It serves as a labeling reagent for the detection and visualization of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Encyclo polymerase mix, by Evrogen (1 mentions)
Micromanipulator mr, by Zeiss (1 mentions)
Sequenase 2, by Thermo Fisher Scientific (1 mentions)
Exo resistant random primer, by Thermo Fisher Scientific (1 mentions)
Klenow buffer, by Thermo Fisher Scientific (1 mentions)
Klenow fragment, by Thermo Fisher Scientific (1 mentions)
Cy3 and cy5 fluorescent dyes, by GE Healthcare (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Random primers dna labeling system, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Axioimager zi microscope, by Zeiss (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
6 protocols using tamra 5 dutp
1
Microdissection and Amplification of Sex Chromosomes
2
Random Primer Labeling of DNA Probes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA probe obtained by microdissection procedures was labelled in a DOP-PCR reaction in the presence of TAMRA-5-dUTP as described earlier [23 (link)]. Fragments obtained by gene-specific PCR were labelled using a Random Primer Labelling protocol: 25 μl of labelling reaction contained 50 ng DNA, 1× Klenow buffer (Thermo Fisher Scientific), 44 ng/μl Exo-Resistant Random Primer (Thermo Fisher Scientific) 0.1 mM dATP, dGTP, dCTP, and 0.015 mM dTTP, 0.016 mM TAMRA-5-dUTP, or Biotin-11-dUTP (Biosan, Novosibirsk, Russia), and 5 U of Klenow fragment (Thermo Fisher Scientific TM) in a PCR tube. The required amounts of DNA, Klenow buffer, and Random Primers were mixed, brought up to 12 μl with water, and heated at 95 °C for 5 min in a thermocycler. The solution was chilled on ice, and appropriate amounts of nucleotides, Klenow fragment, and water were added to reach 25 μl. The reaction mix was incubated at 37 °C for 18 h. Fluorescence in situ hybridization (FISH) was performed using a previously described standard protocol [25 , 27 ].
3
Fluorescent dsDNA Labeling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As a TAMRA-dsDNA probe, the human Alu repeat labeled with TAMRA-5′-dUTP (Biosan, Russia) using PCR was used (Dolgova et al., 2014 (link)). 5 × 105 cells were exposed to 0.1 μg of TAMRA-dsDNA in 500 μl of appropriate medium without additives for 30 min in the dark at room temperature. Cells were then subjected to a single washing with the appropriate medium and resuspended in the desired volume of the medium or phosphate-buffered saline (PBS) for further assays.
4
Fluorescent In Situ Hybridization of Anopheles Genome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene-specific primers were designed to amplify unique exon sequences from the beginning and end of each scaffold using the primer-BLAST program (Ye et al. 2012 (link)) available at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ). The primer design was based on gene annotations from the AalbS1 genome assembly available at VectorBase (https://www.vectorbase.org/organisms/anopheles-albimanus/stecla/aalbs1 ) (Giraldo-Calderon et al. 2015 (link)). PCR was performed using 2X Immomix DNA polymerase (Bioline USA Inc., MA) and a standard Immomix amplification protocol. Amplified fragments were labeled with Cy3 and Cy5 fluorescent dyes (GE Health Care, UK Ltd, Buckinghamshire, UK and Enzo Biochem, Enzo Life Sciences Inc., Farmingdale, NY) or TAMRA-5-dUTP (Biosan, Novosibirsk, Russia) using a Random Primers DNA Labeling System (Invitrogen, Carlsbad, CA). FISH was performed according to the previously described standard protocol (Sharakhov 2015 ). DNA probes were hybridized to the chromosomes at 39° during 10–15 hr in a hybridization solution (50% formamide; 10% sodium dextransulfate, 0.1% Tween 20 in 2XSSC, pH 7.4). Chromosome preparations were washed in 0.2XSSC (saline-sodium citrate: 0.03 M sodium chloride, 0.003 M sodium citrate) and counterstained with DAPI in ProLong Gold Antifade Mountant (Thermo Fisher Scientific Inc.).
5
Fluorescent Alu DNA Labeling and Cellular Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was labeled with TAMRA-5’-dUTP (N-90100, Biosan, Novosibirsk) using PCR. The PCR template was human Alu repeat material cloned in pBlueScript SK(+) (Alu-pBS), this repeat encompassing the tandemly repeated AluJ and AluY sequences (NCBI: AC002400.1, 53494–53767). Standard M13 primers were used for amplification. PCR purification was done by standard phenol-chloroform extraction followed by ethanol precipitation using ammonium acetate as a salt. The quantity of eDNA being added to the cells (Alu-TAMRA DNA, pUC19 (#440060, Medigen, Novosibirsk), pEGFP-N1 (#6085-1, Clontech), sonicated pEGFP-N1) was 1 μg plasmid DNA/106 cells and 0.2 μg Alu-TAMRA DNA/106 cells. The cells that incorporated the fluorescently labeled DNA probe were analyzed by either FACS (BD FACSAria, Becton Dickinson) or by fluorescence microscopy (laser scanning microscope LSM 510 META (Zeiss), ZEN software or AxioImager ZI microscope (Zeiss), ISIS software).
6
Salivary Gland FISH on Polytene Chromosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Salivary glands of third instar larvae were dissected in Ephrussi–Beadle solution and transferred to a small glass cup with a mixture of ethanol and acetic acid (3:1). After 20 min fixation at room temperature, salivary glands were squashed in 45% acetic acid, frozen in liquid nitrogen, and stored in 70% ethanol at −20 °C. Fluorescence in situ hybridization (FISH) on polytene chromosomes was performed as described in [48 ]. Thirty-five DNA probes were obtained by standard PCR and then labeled with Flu-12-dUTP or Tamra-5-dUTP (Biosan) in random-primed polymerase reaction with the Klenow Fragment. Table S1 lists probes used in this study. Chromosome squashes were analyzed while using epifluorescence optics (Olympus BX50 microscope) and photographed with CCD Olympus DP50. For every probe colocalization, at least 50 nuclei on several slides were analyzed.
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!