Construction of the MsParA (Ms6939) knockout strain of M. smegmatis and Southern blot assays.(A) Schematic representation of the recombination strategy for the
Gene replacement strategy and Southern blot analysis of progeny from heterozygous crosses. (a) The wild-type P-selectin allele, the replacement vector, and th
Southern blot definition is - a blot consisting of a nitrocellulose or nylon sheet containing spots of DNA for identification by a suitable molecular probe.
Restriction digest, also known as DNA fragmentation, is a method to cut DNA into smaller fragments of interest with restriction enzymes. In our experiment, we performed double digests which involves cutting the vector and insert DNA with two different restriction enzymes. XbaI and PstI restriction enzymes are used to cut the insert, in this case Green Fluorescent Protein (GFP). SpeI and PstI are used to cut the plasmid (ROO40 and R0010). GFP is designated as the insert because it is much larger (approximately 700 bp) than R0040 or R0010. If R0040 or R0010 were to be used as inserts, they would not show up clearly during the gel electrophoresis. It is important to use different restriction enzymes to ensure correct insert orientation during ligation with the vector. Things to take into account when performing a restriction digest: (1) Buffer compatibility: When performing a single digest, a buffer with a low salt concentration should be used first followed by a buffer with a higher salt ...
Yng Chen writes: , In Southern hybridization experiments how important is hybridization , temperature? What are the effects of rasing or lowering the hybridization , temperature? One generally hybridizes 15-20 C below the Tm to get favorable kinetics. So the exact temp. is not critical with respect to enforcing stringency. The only exception is if youre trying to do something at very low stringency, then you may need to lower the hybridization temp. below standard conditions. , How does one determine the proper washing conditions (assuming 100% , match in base paring). [for oligo probes] , If you get a weak signal and you know that you have a lot of DNA , on the membrane and that you have good specific activity on your , probe, how do you know if this is because your washing conditions , are too strigent or that the signal is non-specific. You start with a calculation, and then confirm by doing a temperature series against a cloned control target. If youre trying to discriminate a one base ...
Generation of ACE2-deficient mice. BAC145d21 containing a portion of the murine Ace2 gene was identified by Southern hybridization using a 1.8-kb EcoRI fragment of the human ACE2 cDNA. A 5.2-kb fragment of BAC145d21 that included the exon containing sequences encoding the active site of the ACE2 enzyme (+1069 to +1299) was subcloned into the yeast shuttle vector YCpLac22 (pMD44). A 3.4-kb fragment containing the NEO/URA3 cassette from pRAY-1 and flanked by Ace2 genomic sequence was generated by PCR and cotransformed with pMD44 into yeast strain YPH501 for homologous recombination. The final linearized targeting construct, shown in Figure 1A, was electroporated into MPI1-12D ES cells that had been derived from 129/SvEvfBRTac mice. G418-resistant ES cells were screened for homologous recombinants by Southern blot hybridization with genomic probes that were outside of the targeting construct (Figure 1, A and B). Targeted ES clones were then injected into C57BL/6H blastocysts to generate chimeras. ...
Dear Netters: I am screening A. thaliana genomic library with a heterologous probe. By screening about 160000 plaques(!), I have got 9 independent clones. These clones are purified to homogeneity by 3-5 time screening. All washes were to high stringency. To double check that these (or at least one of them) are corresponding to the right gene and not other related genes (i.e. other members of a gene family), I do the following test primarily. That is, I made a southern blot of A. thaliana genomic DNA digested by 3 different restriction enzymes. Then, I hybridize the original probe (which was used for screening) and the probe made from the whole insert (9-17 Kb) of the isolated clones. I expected that the banding pattern of the S. blots of the isolated clones to be similar (or include) that of the S. blot probed with the screening probe. This did not happened for any of the isolated clones. Any advice or comment regarding this problem is greatly appreciated. Thanks. Ali My E-Mail Address is: ...
P{y[+t7.7]=3wHy} associated with end of Df; Hobo element may be mobilized if crossed to H strain, B.G. Cytological breakpoints estimated from molecular breakpoints, K.C ...
Important Information about Clavam 625 - an Antibiotic medicine. Why it is prescribed, dosage and possible side-effects of Clavam 625
Hello SIR, Thanks for your reply. Today is 20th day and still my urologist kept me on half dose of clavam 625 and levoflox 250 twise a day for another 5 days.. ...
Southern blotting is an experimental procedure where DNA, from a genomic or other source, is digested with a restriction enzyme and then separated by size using gel electrophoresis. The fragments are transferred from the gel onto a membrane (blotted) which is then incubated with a labelled single-stranded DNA probe. Such a procedure allows one to locate a particular sequence of DNA within a complex mixture of DNA. From a gene targeting perspective, Southern blotting can be used to detect whether a targeting event has successfully taken place. Designing a good Southern blot probe for a particular gene or locus involves finding a stretch of DNA sequence at that locus, generally 500-1000bp long, that has the desirable qualities of being unique to that locus, with little or no repetitive DNA content. Molecular biologists tend to design their probes manually, by excising portions of genome sequence from online genome browsers (such as such as Ensembl) and then pasting them into a genome-search ...
Revision as of 14:33, 2 October 2019 by Webref (talk , contribs) (Created page with A procedure in which DNA restriction fragments are transferred from an agarose gel to a nitrocellulose filter, where the denatured DNA is then hybridized to a radioactive prob...) ...
(KudoZ) Russian to English translation of блоттинг-гибридизация: blot hybridization [Biology (-tech,-chem,micro-) (Medical)].
Genetic information processingMobile and extrachromosomal element functionsPlasmid functionsentry exclusion protein TrbK (TIGR04361; HMM-score: 9.9) ...
DIG southern blot - posted in Molecular Biology: Please can someone give me some suggestions as to why my DIG SB is not working. Only the DIG-labeled ladder and positive control work. I have tried different DNA concentrations, less stringent hyb/washes. I know this protocol should work as it works for people in another country working on the same plant. I used the same probe and followed their protocol. the only difference is the TNA extraction (I use CTAB Doyle and Doyle), and NBT instead...
Southern blot analysis indicated the presence of a single bar gene flanked its regulatory genetic elements. further analysis indicated the the 3 nos terminator was truncated during the transformation process. Additionally a second copy of the 35S promoter or part of it was inserted into the host genome ...
Hi, everyone;. I have done a series of PCR and found that there is slight difference between control and treated group according to bands stained with EB in 2% agarose gel. I was told that such a result is much less sensitive than that of southern. So I blotted such gel to membrane for further southern analysis. Here is the question, if I can use the same PCR product to construct my probe? Yes or No? and How ...
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The aim of this study was to demonstrate that good characterization of S. cerevisiae clinical isolates can be achieved by using a molecular method such as Southern blot hybridization analysis. Even though the development of a molecular typing method useful for the characterization of a yeast infection is a desirable goal for many mycologists, the number of studies in which the epidemiology of S. cerevisiae infections has been investigated is very limited (9, 17, 34). A universally accepted typing method for this organism has been not defined. This could be due in part to the low prevalence of diseases caused byS. cerevisiae, particularly of vaginal infections. However, in our investigation, these infections have been of considerable interest. As described above, of 513 women who visited our Microbiological Service in the study period, 5.8% had S. cerevisiae in their vaginal swabs. This was in agreement with the data reported by Agatensi et al. (1), by which the incidence of vaginal infections ...
The deletion mutation was introduced into the ABI 2.1 embryonic stem (ES) cell line (Soriano et al., 1991) and transmitted to the germline as described previously (Bullard et al., 1996). Chimeric mice were bred with C57BL/6J mice, and the mice used in these studies were maintained on a mixed 129/SvEv and C57BL/6J background. The mutation is being back-crossed on the C57BL/6J background for future work.. Southern, Northern, and Western analyses. Southern blot hybridization was performed according to standard methods (Sambrook et al., 1989) using a hybridization solution of 0.125m NaPO4, pH 7.0, 0.25 mNaCl, 1 mm EDTA, 10% polyethylene glycol (PEG-8000), 7% SDS, and 1% bovine serum albumin (BSA) at 65°C overnight followed by washing to a final stringency of 0.2× SSC/0.1% SDS at 65°C and autoradiography at −80°C.. Total RNA was isolated from brain after homogenization in Ultraspec II (Tel Test, Houston, TX). Total RNA was resolved on a 1.2% agarose gel in 10 mm NaPO4 buffer, pH 6.8, after ...
Genetic information processingMobile and extrachromosomal element functionsProphage functionsputative phage terminase, small subunit, P27 family (TIGR01558; HMM-score: 13.7) ...
OBJECTIVES--To determine the prevalence of urethral HPV infection, as indicated by the presence of HPV DNA in semen, in males with and without penile warts. DESIGN--Prevalence study of HPV types 6/11 and 16 DNA using PCR and Southern blot hybridisation analysis of semen. SETTING--Department of Genitourinary Medicine, Blundell Street Clinic, Leeds General Infirmary and the Assisted Conception Unit (ACU) Kings College, London. SUBJECTS--Patients attending the Genitourinary Clinic for treatment of sexually transmitted diseases including penile warts and males attending Kings ACU for investigations of infertility. MAIN OUTCOME MEASURES--HPV DNA detected by polymerase chain reaction (PCR) and/or Southern blot hybridisation in semen. RESULTS--HPV DNA was detected by PCR in 23 of 27 (85%) specimens from patients attending the GUM clinic for treatment of genital warts and in one of two specimens from patients attending the clinic for other conditions. By Southern blot, nine (33%) of the 29 specimens ...
We present data demonstrating the gene expression of substance P and its receptor in human peripheral blood-isolated monocytes and macrophages. Using the RT-PCR assay, preprotachykinin-A (substance P) mRNA is detected in human peripheral blood-isolated monocytes and macrophages. Among the alpha, beta, and gamma transcripts of the substance P gene, only the beta and gamma transcripts are detectable in these cells. By Southern blot assay these RT-PCR-amplified transcripts are recognized using a specific substance P probe. Sequence analysis of the RT-PCR products from both monocytes and macrophages also confirmed the structure of these transcripts, which are identical to those found in human neuronal cells. At the protein level, both human monocytes and macrophages produced endogenous substance P as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released substance P from both human monocytes and macrophages. In addition, using nested RT-PCR ...
Transgene vector and generation of transgenic mice.We obtained the Crx genomic clone from a 129SVJ mouse library (Stratagene, La Jolla, CA) by using a mouse Crx cDNA probe. We ligated and subcloned a 10 kb XhoI (partial digestion)-EcoRI fragment and a PCR-amplified 2 kbEcoRI-SmaI fragment containing exon 1 into a pβ-gal-Basic vector (Clontech, Palo Alto, CA) to make the Pcrx12k-lacZ construct (see Fig. 1A). Sequencing verified the 2 kb EcoRI-SmaI fragment. The Pcrx2k-lacZ vector contains the 2 kbEcoRI-exon 1 fragment and a 10 kbSmaI-EcoRI fragment that contains the first intron.. We extracted the Pcrx2k-lacZ and the Pcrx12k-lacZ from the recombinant plasmids by aNotI and SalI digestion. We fractionated theNotI-SalI fragments by electrophoresis on a 0.8% agarose gel and purified them by electroelution in dialysis tubes. We microinjected the DNA fragment into pronuclei of B6SJL/F2 C57BL/6 × SJL F2 hybrids. Then Southern blot hybridization of a HindIII-ClaI 954 bp fragment of the β-galactosidase ...
This was a hybrid restriction site created during the cloning process (Cap-trapper) for the library creation. Neither of these restriction sites will work to digest the insert from the vector. According to the IMAGE consortium the SalI-XhoI (gtcgag) is located at position 742 of the polylinker sequence (http: //mgc. nci. nih. gov/Vectors/vec_pbluescriptr). Customers can use BamHI (5) and EcoRI (3) to digest out the insert. Other options for sub-cloning are either to use different restriction enzymes lying outside BamHI and EcoRI or to design insert-specific primers based on the insert sequence. You can also find a reference for the cap trapper method here: Carninci et. al. , DNA RESEARCH 4, 61-66 (1997), High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper ...
Nucleic acids from ATCC Genuine Cultures can save you the time and expense of isolating DNA yourself. ATCC offers genomic DNA from well-characterized and authenticated fungal and yeast strains.
Nucleic acids from ATCC Genuine Cultures can save you the time and expense of isolating DNA yourself. ATCC offers genomic DNA from well-characterized and authenticated fungal and yeast strains.
Developmentally programmed deoxyribonucleic acid(DNA) rearrangements are structural reorganisations of the genome that occur reproducibly during the development of a variety of organisms
12404385] The Biosynthesis of Vancomycin-Type Glycopeptide Antibiotics-The Order of the Cyclization Steps This work was supported by the Deutsche Forschungsgemeinschaft (SFB 323) and by a grant of the EU (MEGATOP, QLK3-1999-00650). R. D. S. gratefully acknowledges the support of a Feodor-Lynen Fellowship granted by the Alexander-von-Humboldt Stiftung. We thank Corina Bihlmaier and Volker Pfeifer for help with transformation and Southern hybridization, J. A. Moss (La Jolla (USA)) for critical comments on the manuscript and Prof. Dr. M. E. Maier and Prof. Dr. H.-P. Fiedler (Tubingen) for generous support. (Angew Chem Int Ed Engl. , 2001 ...
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