Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
TY - CHAP. T1 - 5′-Monopyrene and 5′-Bispyrene 2′-O-methyl RNA Probes for Detection of RNA Mismatches. AU - Novopashina, D. S.. AU - Semikolenova, O. A.. AU - Venyaminova, A. G.. PY - 2020/1/1. Y1 - 2020/1/1. N2 - Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5′-monopyrene and 5′-bispyrene conjugates of oligo(2′-O-methylribonucleotides) and their application as probes for fluorescent detection of mismatches in RNA targets.. AB - Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5′-monopyrene and ...
Embryos. Pregnant CD-1 mice (Charles River Laboratories, Wilmington, MA) were killed, and the litters were collected according to the NIH Guide for the Care and Use of Laboratory Animals protocol. Embryos were individually staged according to the method of Theiler (1989).. Probes. The in situ hybridization probe forFng was obtained by screening an 8.5 d postcoitum (dpc) embryonic mouse λgt10 library (a gift from Brigid Hogan) with a mixture of human (Expressed sequence tags, Research Genetics) and chicken Fng (Laufer et al., 1997) cDNAs. Several strongly hybridizing clones were obtained, and one of these, pMFR1, was selected for further analysis. The 2.2 kb insert of pMFR1 contains the entire coding region of murine Fng (Cohen et al., 1997; Johnston et al., 1997). The antisense RNA probe was generated by using T3 RNA polymerase after HindIII restriction digest of pMFR1, and the sense RNA probe was generated by T7 RNA polymerase afterXbaI restriction digest. The NT-3 RNA probe was generated from ...
Parts of the cDNA fragments were ligated into pBluescript II KS vector, and we used this plasmid to synthesize a Dig (digoxygenin)-labeled RNA probe. Dig-labeled RNA probes were made by in vitro transcription from the linearized plasmid with a Dig RNA labeling kit (Roche). T7 or T3 RNA polymerase (Roche) was used in the in vitro transcription to synthesize an antisense RNA probe for hybridization.. In each step, it took 15 min to form a drop of dicyemids in the bottom of each microtube. Dicyemids were washed 5 times and then fixed overnight at 4°C in 4% paraformaldehyde in 0.5 M NaCl and 0.1 M MOPS buffer. After fixation, the dicyemids were dehydrated in an ethanol series (30%, 50%, 70%) for 10 min each and the dicyemids were stored in a 70% ethanol up-series at -30°C. Dicyemids were hydrated in an ethanol down-series (70%, 50%, 30%) for 5 min each, and were washed 3 times in PBT (phosphate buffer saline [PBS] with 0.1% Tween 20). The hydrated dicyemids were partly digested by protease K (0.5 ...
mutaFISH™ EGFR T790M T790wt RNA Probes is designed to detect human EGFR T790M gene mutation on single strand RNA in cells using in situ rolling-circle amplication technology. (FP0001) - Products - Abnova
In article ,4dgqf7$10g0 at majestix.uni-muenster.de, Torsten Boerchers ,borcher at uni-muenster.de, writes: ,Torsten Boerchers ,borcher at uni-muenster.de, wrote: ,,Forwarded from rhubner at molbiol.ox.ac.uk: ,, ,,hello Torsten, ,, although you dont give details about those multiple bands I would ,,suspect either premature termination or/and read-through (not completely ,,linearized... ,,who can COMPLETELY linearize plasmids?) of the T7 pol beyond the RE ,site , ,,,multiple bands depending on run-on transcription intensity... ,,wouldnt cutting with a second RE help troubleshooting? but, what ,,then ? (i.e., how to stop such rolling-circles efficiently?). In the ,case of premature termination I fear nothing special can be done... ,,oh yes, you might also have alternative initiation (change into other ,RNA pol vector to check? can be predicted by seq analysis to some ,extend...) ,, Obviously _I_ too need to read/hear/experience more about T7 pol ,,transcription initiation/termination... any ...
so what are you making the probes for, and what kind of probes are they? because if your insert isnt HUGE, you can always PCR the insert out (using primers that anneal outside of the T7 and Sp6 sites so that they are included in your PCR product), then use the PCR product for your transcription reaction. I do this to make T7 and Sp6 RNA probes for in situ hybridisations and it works really well.... ...
Digoxigenin (DIG)-labeled RNA antisense probes are widely used for in situ hybridization due to their high sensitivity and specificity. DIG-labeled RNA probes are also stable for more than a year, making them ideal for long-term studies with high consistency and low technical variation. In this application note read how DIG-labeled probes were used to investigate the localization of human hepatocyte growth factor (hHGF), a cell surface cytokine binding the c-MET-receptor, implicated in the tyros
The RNA probe was in vitro transcribed with the recombinant of the Y-chromosome specific deoxyribonucleic acid (DNA) sequence and transcription-vector Bluescript SK M13+. The preparation of the RNA probe is not difficult, but it does require a subcloning procedure which is unnecessary in the preparation of the DNA probe. The results indicate the superiority of the RNA probe over the previously studied DNA probe. The RNA probe has a high hybridization efficiency which increases the sensitivity of the test and shortens the period of time necessary for obtaining the resulting stains. 2 figures, 7 references. (Author abstract modified) ...
This protocol is designed for In Situ Hybridization and colorimetric detection of digoxigenin-labeled RNA probes on paraformaldehyde fixed cryosectioned tissue. This protocol has primarily been tested on mouse embryos and on brain tissue and takes approximately three days. ...
The ribonuclease protection assay (RPA) is a highly sensitiveand specific method for the detection and quantitationof mRNA species. The assay was made possibleby the discovery and characterization of bacteriophageencodedDNA-dependent RNA polymerases (SP6 T7and T3).,RNase,Protection,Assay,(RPA),,,Using,DIG-Labeled,RNA,Probes,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology
The ability to visualize the expression of a gene in both time and space is an essential tool of developmental biology. Here, we detail a robust method for in situhybridization of RNA probes to...
Traces of DEPC modify purine residues (A+G) in RNA by carboxymethylation. Therefore, DEPC must always be removed from solutions or containers by autoclaving or heating at 100°C for 15 min. Cell-free translation of carboxymethylated RNA will yield less protein than with unmodified template. Hybridisation is not seriously affected unless the RNA probe is heavily ...
5-(3-aminoallyl)uridine 5-triphosphate (AA-UTP) is a cost-effective alternative to direct incorporation of a labeled-UTP when preparing non-radioactive RNA probes by in vitro transcription. AA-UTP is a good substrate for T7, T3 and SP6 RNA polymerases to
IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybrid
Kit Component:- KN207072G1, BRUNOL5 gRNA vector 1 in pCas-Guide vector- KN207072G2, BRUNOL5 gRNA vector 2 in pCas-Guide vector- KN207072D, donor…
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene. Since James Watson and Francis Crick revealed the double helix nature of DNA molecule(Watson & Crick, 1953), the hydrogen bonds between the four bases are well known: adenine always binds to thymine and cytosine always binds to guanine. This binding pattern is the basic principle of modern genetic technologies. Joseph Gall and Mary Lou Pardue published a paper in 1969 demonstrating that radioactive marked ribosomal DNA can be used to detect its ...
Purpose: : Musashi (MSI), an mRNA binding protein, has been shown to be expressed in neural progenitor cells. (reviewed by Okano et al., 2005) The goal of this study was to determine the expression of MSI in the chick retina, with special emphasis on putative stem cells within the retina. Methods: : Immunohistochemistry and in situ hybridization were performed with 14 um sections of E3, E4, E5, E6, E8 and E18 eye using anti-MSI antibody and a MSI specific Digoxigenin-labeled RNA probe. Results: : MSI appeared to be expressed in the presumptive amacrine and photoreceptor cells of the developing retina, the ciliary margin zone where stem cell preside, and in the lens epithelium. Conclusions: : MSI labels more than stem cells in the chick retina. ...
Tissue preparation. Twenty adult male Sprague Dawley 20 rats (100-120 gm body weight) were anesthetized with choral hydrate (3.5 mg/100 gm body weight) and perfused transcardially with a solution of 4% paraformaldehyde in 0.1 m phosphate buffer (PB), pH 7.3. Brains were post-fixed overnight, rinsed with PB, and sequentially transferred to 12%, 14%, and 16% sucrose solutions. Brains were then frozen on dry ice, and sections of 30-40 μm thickness were obtained on a cryostat.. Probe preparation. [35S]- and [33P]-labeled sense and antisense RNA probes were generated from a 730 basepair (bp) PstI insert (corresponding to nucleotides 1500-2230 of the rat 5HT3R cDNA), using the TransProbe T Kit (Pharmacia, Piscataway, NJ). The [35S]- and [33P]-labeled antisense probes were synthesized separately.. In situ hybridization-immunocytochemistry labeling. In situ hybridization combined with immunolabeling was performed as described previously (Morales et al., 1996a). Free-floating cryosections were incubated ...
Hi all. Is there anybody who could advice me a reliable protocol for whole mount in situ hybridization with organs dissected from the last instar larva, e.g. salivary , gut, Malpighian, etc. ? I am especially interested in the fixation step and necessity and extend of proteinase treatment. I am using DIG RNA probe. Please respond directly by e-mail to mach at entu.cas.cz. Thanks for your help. Vaclav Mach Institute of Entomology Czech Republic ...
THOMAS:. Wash. Rock. Aspirate. Repeat.. Six weeks of diligent work had culminated with six delicately miniscule mouse embryos, so transparent that my eyes often lost track of them as they sloshed around in solution. My mentor and I had spent the past month running PCRs, conjuring up gels, and transforming plasmids into bacteria, all in preparation for this final step: the in situ hybridization. The purpose of this complicated sounding process was to insert RNA probes into the embryos, which would allow us to observe the expression pattern of the gene Fgf12 in embryonic heart development (specifically in the atrium). The next four days would be filled with myriads of strange solutions comprised of mysterious, unpronounceable substances. These deceptively clear liquids were capable of performing painstaking chemical processes at the molecular level, something that my mind could hardly fathom. Each step involved washing the embryos with different solutions (which could contain anything from ...
Fig. 5 pax-6 is expressed in limited regions of the neural keel. Paraffin sections of embryos were hybridized with 35S-labelled RNA probes (probe A, probe C gave the same results). (A,B) Parasagittal sections of a 14 h embryo. Expression is seen in the optic vesicle (A, B), the prospective forebrain (B) and the prospective hindbrain and spinal cord (A, B). Lines in B indicate the plane of section in panels C and D. (C) Cross-section of the prospective forebrain at 14 h. Expression is seen in the dorsal half, excluding the dorsal-most cells of the forebrain. (D) Cross-section of the neural keel at 14 h. Expression is seen in the ventral half. (E) Parasagittal section of the optic vesicle (ov) at 14 h. Expression is seen in the ventral and lateral part of the vesicle. Little or no expression is detectable in the dorsal part. Expression is also seen in the ectoderm (ect) overlying the optic vesicle. (F) Horizontal section of an 18 h embryo. Expression is seen in the diencephalon (di) and the ...
Adding T7 promoter sequence to vector - posted in Molecular Biology: Hi all:I am trying to make RNA probe for ISH. I ordered cDNA clone from a company and I realized that the vector they used does not have T7 or T3 or SP3 promoter sequence. So, how can I add the promoter sequence to my vector? Also, I streaked the glycerol stock which I got yesterday but no colonies today, what could have gone wrong?Vector:pME18S-FL3Thanks in advance
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We have investigated the usefulness of serum hepatitis delta virus (HDV) RNA detection using a slot hybridization analysis of serum samples from ten patients with acute hepatitis and delta markers (group I), from 28 patients with chronic delta hepatitis (group II) and from seven liver graft recipients with hepatitis B virus (HBV) and HDV related cirrhosis or fulminant hepatitis (group III). The slot-blots were hybridized with both HDV-complementary DNA and single-stranded RNA probes. With the single-stranded RNA probe, HDV RNA was detected in the first serum sample available in 9/10 of the patients with acute hepatitis (group I). In addition, HDV RNA was detected in 8/9 and 7/8 of the samples obtained within and after 1 month of the onset of hepatitis. Five of the ten patients scored positive for HDV RNA and negative for hepatitis delta antigen (HDAg) while one was negative for HDV RNA and positive for HDAg. The same RNA probe enabled the detection of serum HDV RNA in 21/28 chronic hepatitis ...
... products include RNA purification kits, RNase protection (RNasin RNase Inhibitor), RNA quantification dyes and fluorometers, and RT-PCR kits. We also offer systems for RNA synthesis (in vitro transcription), including production of RNA probes (Riboprobes).
Array Hybridization and Detection of Fluorescence was performed as follows. 12 ng of IVT was fragmented in 40 mM Tris-actetate, pH 8.0, 100 mM potassium acetate, and 30 mM magnesium acetate for 35 minutes at 94 °C. The fragmented, labeled RNA probes were diluted in hybridization buffer at a final composition of 1 x 2-NMorpholinoethanesulfonic acid (MES (buffer (pH 6.5), 50 pM Bio948 (control biotinylated oligo that hybridizes to landmark features on the probe array (Genetics Institute, Cambridge, MA)), 100 u.g/ml herring sperm DNA (Promega, Madison,WD, 500 u.g/ml acetylated BSA (Invitrogen Life Technologies) and 1 jul/jag standard curve reagent (Proprietary reagent supplied by Gene Logic, Gaithersburg, MD). This hybridization solution was pre-hybridized with two glass beads (Fisher Scientific, Pittsburgh, PA) at 45 °C overnight. The hybridization solution was removed to a clean tube, heated for 1-2 min at 95 °C and microcentrifuged on high for 2 minutes to pellet insoluble debris. Affymetrix ...
Definition : Molecular assay reagents consisting of a known fragment of nucleic acid, typically labeled with a tag. Probes may be classified according to their chemical structure as DNA or RNA probes, strandedness as double- or single-stranded (ds and ss, respectively), and origin as cloned (recombinant), genomic, or synthesized (oligonucleotides). Probes may be radioactively or nonradioactively (both enzymatic and nonenzymatic) labeled. Nucleic acid probes are used in tests to obtain information about the genetic characteristics of a sample; these procedures are generically known as nucleic acid assays and include hybridization assays, amplification methods (e.g., polymerase chain reaction), and electrophoretic separation.. Entry Terms : "Probes, Nucleic Acid" , "Nucleic Acid Probes" , "Reagents, Molecular Assay, Nucleic Acid Probe". UMDC code : 19666 ...
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The ability of labeled DNA or RNA probes to bind with high affinity and specificity to complementary nucleic acid sequences forms the basis of nucleic acid hybridization assays. Immunoassays, on the other hand, exploit the strong and specific interaction between antigens and antibodies. One aspect of this dissertation deals with enhancement of the sensitivity of bioluminescence hybridization assays based on the photoprotein aequorin. This is achieved by introducing, enzymatically, multiple aequorin labels per DNA hybrid. The bound aequorin is determined by its characteristic bioluminescence. An 8-fold improvement in sensitivity is observed with the amplified assay as compared to an assay without the amplification step. Another aspect of this dissertation involves the development of two novel microtiter well-based DNA hybridization assays in which an expressible cDNA fragment encoding firefly luciferase serves as a reporter molecule. The reporter molecule undergoes in vitro expression through coupled
We plan to apply the methodologies developed from our current NMFS-funded project for mtDNA markers, DNA fingerprinting, RNA probes and PCR technologies to quantify low-levels of Perkinsus marinus infections in order to fully describe the etiology of this oyster disease. Our progress in working with genetic markers of P. marinus has: Developed a sensitive PCR-based diagnostic assay for detecting P. marinus infections ; Identified an extrachromosomal relic plastid-genome in P.
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TY - JOUR. T1 - Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. AU - Melton, D. A.. AU - Krieg, Paul A. AU - Rebagliati, M. R.. AU - Maniatis, T.. AU - Zinn, K.. AU - Green, M. R.. PY - 1984/9/25. Y1 - 1984/9/25. N2 - A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific ...
Figure 4. Localization of GRK7 mRNA in ground squirrel retinas using in situ hybridization.. 35S-labeled antisense and sense RNA probes were produced from the 146-bp fragment corresponding to nucleotide sequence 1019-1164 by in vitro transcription and hybridized to cryostat sections of ground squirrel retina as described in the "Methods". (A) Dark field image of ground squirrel retina hybridized with an antisense probe for GRK7. Retinal pigment epithelium (RPE), photoreceptor cell layer (PCL), inner segment layer (INL), ganglion cell layer (GCL). (B) Dark field image of ground squirrel retina hybridized with a sense probe for GRK7. Abbreviations are as described in (A). Since the retinal pigment epithelium is black, it appears white in dark field images.. ...
Samples Cervical swab specimens, which were obtained for screening test of cervical cancers, were collected from a total 146 women at Seoul St. Marys Hospital between January and December of 2011. All specimens were collected, placed in a ThinPrep PreservCyt solution (Hologic Inc., Malborough, MA, USA) and a SurePath solution (BD, Franklin Lakes, NJ), and were then stored at -70°C until assayed for HPV tests. All specimens were classified into 4 groups according to their cytologic results: negative (n=62), ASCUS (n=39), LSIL (n=35) and HSIL (n=10).. HC2 test HPV DNA testing by the HC2 test was performed with an HC2 assay system according to the manufacturers protocol (Qiagen Inc.,Valencia, CA, USA). The specimens were denatured at 65°C for 45 minutes and hybridized under high-stringency conditions with a mixture of RNA probes that detect 13 different oncogenic HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The resultant DNA-RNA hybrids were captured on the surface of the ...
[email protected] wrote: Hello Histonetters, I would like to do fluorescence in situ hybridization on paraffin embedded sections using RNA probes. I would appreciate it very much if anybody who has worked on this could share the protocol with me. Thanks in advance. Agripina Agripina C. Suarez Cardiovascular Research Laboratory McDonald Research Wing, St. Pauls Hospital 1081 Burrard St., Vancouver B.C., Canada V6Z 1Y6 Phone: (604) 631-5659 Fax: (604) 631-5351 Hi Agripina, I would suggest the Hyb-Probe system from Shandon Lipshaw Cat.#502100 this system utilizesFluorescein-labeled nucleic acid probes. This system allow identification of specific nucleic acids DNA and RNA. This system is very time efficient and sensitive. Good luck and I Hope this info. helps. Sincerely, Carol Tucker-Burden Emory University Atlanta, GA. Dept. Of Surgery Transplant Immunology Research Ctr. ____________________________________________________________________ More than just email--Get your FREE Netscape ...
In this report, we show that the products of UL47, UL49, and US11 ORFs bind RNA in vitro and in the context of infected cells, and that the packaged RNAs can be expressed in infected cells. We also show that VP22, the product of the UL49 ORF, mediates the transfer of the RNA from cell to cell. Relevant to our results are the following:. (i) The procedure we have used to identify the protein capable of binding RNAs was to electrophoretically separate virion proteins in denaturing gels, renature the proteins in situ, and react them with a labeled riboprobe representing the RNA detected in all virion preparations tested. Using this procedure, we unambiguously demonstrated that three virion protein bands bind RNAs. These proteins were identified as the products of the UL47, UL49, and US11 genes. In these assays, we used as probe the most abundant RNA packaged in virions. Because we used a riboprobe representing a single viral RNA, we cannot exclude the possibility that there exist virion proteins ...
Expression of Raldh1. In situ hybridisation was performed on wholemount embryos with DIG- or fluorescein-labelled riboprobes. Unless otherwise stated, rostral i
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Labeled FISH probes for identification of gene split using Fluoresecent In Situ Hybridization Technique. (Technology) (FS0052) - Products - Abnova
原位雜交技術(In Situ Hybridization, ISH)是結合分子生物學、細胞學以及組織化學的一門新興技術。此技術始於1969年,耶魯大學的Gall等人首先將爪蟾核醣體基因探針與其母卵細胞
Probe definition is - a slender medical instrument used especially for exploration (as of a wound or body cavity). How to use probe in a sentence. Synonym Discussion of probe.
I just got accepted to CRNA school. My question is I was always told a benfit to being a CRNA is you are able to do anesthesia with nurses hours. Is this true. Can I be a CRNA and work 36-43 hrs a week and still make 6 figures?
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What did everyone think of the interview? It was not as crazy as I expected. Few drugs and personal questions. Good luck and hope to see you in the fall!!!