All assays use the same standardized protocol to generate results in three easy steps. Nothing beats the excitement and sense of pride that accompanies a successful experiment. And while good science is certainly the driving force behind these accomplishments, the ability to maximize your productivity and opportunities for success should not be ignored. We all can relate to those awkward time gaps when your gel is running or your samples are washing or incubating.
In this article, we will list our top 10 tips for maximizing your lab productivity. Designed for an accelerated workflow, the MagNA Pure 96 System is optimized for high throughput nucleic acid purification — 96 samples extracted in less than one hour - maximizing productivity and dramatically reducing handling errors. In this article, we will discuss the importance of starting with a high-quality cDNA template for your RT-qPCR experiments and our top tips for how to do so.
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A compilation of publications about Roche Genomic products specific to your research applications. Online Technical Support Find help. Last Update On A photocopy or photograph of the result may be made as a. The chromogenic detection procedures and the time required for each stage are listed in.
Unless otherwise indicated, all of the following incubations are per for med at room. If you are processing smaller blots e. Be for e you start: First prepare the working solutions for the detection procedure:. Washing Buffer 1 0. If you let the membrane dry at any stage of the prehybridization, hybridization, or detection. Always prepare the stripping buffer just be for e use. With this technique, we have successfully.
Section 3 of this chapter described how DIG -labeled probes could be used to fi nd target. This section describes how to use DIG -labeled probes to. Many are available from Roche Applied Science catalog numbers given below. Hybridization bags 11 Plastic bags fi tted with a spigot, which facilitates. The procedures for colony and plaque screening and the time required for each are listed. An estimate of the time required for each procedure is also given. Be for e you begin: For the procedure below, determine the appropriate hybridization.
If you are using a different hybridization buffer , see Chapter 5 A for instructions on. To avoid contamination and background for mation, always use gloves and a pair of. If you are processing more than one disc, increase the. These steps may. For one mm disc,. If you are processing more than one disc, increase the volumes until there. Two sets of M13 phage clones. The fi rst set contai ned the gene for ampicillin resistance Amp r ; the second contained the gene. Aliquots of these two sets were mixed and plated on a lawn of E.
The plaque lift was screened with a mixture of two probes, each pre sent at The fi rst probe was a DIG -labeled probe complementary to the Amp r gene; the second. Probe-target hybrids were detected in two stages,. Between the. The visible plaques. Panel B shows the result of the second stage detection with streptavidin-AP, which recognizes.
Since the membrane was not stripped or the AP inactivated between the stages, the visible. The Tet r clones can be identifi ed by comparing.
Almost bacterial colonies were positioned on a membrane at high density in a grid pattern. The colony. Data courtesy of Dr. Bouchier, Genethon, France.
Result: There are only two differences in the two screens. First, the detection took 18 h with the radioactive probe,. Second, two additional clones are.
Note that the exposures in both panels have been adjusted to show the gridlines of the colony pattern, which. Array screening is also used to detect differences in. Yeast RNA 10 Carrier nucleic acid for the hybridization buffer. The procedures required for cDNA screening are summarized in the fl ow chart below. For details, see the package insert for the product, which is available on our Web site. For detailed de scriptions of differential screening procedures. Roche Applied Science offers a wide range of products for nonradioactive analysis.
In this chapter, we briefl y describe some of these nonradioactive assays. For detailed protocols,. Chemiluminescent detection has become the method of choice for detection of proteins. It has the added advantage. Determinations of telomere length may provide important in for mation about normal.
For example, accelerated. Such assays may be useful, for example, in studying the development of. DIG -labeled hybridization probe, specifi c for telomeric.
Assay, Cat. It is based on the differential. Kit, Cat. Reagents are included in DIG. The reaction was specifi c because the protein could be competed off the labeled oligonucleotide by increasing.
See Section 6. This allows low level DIG labeling without signifi. This level of DIG labeling. Labeling mix Panel A. Each probe was bp and recognized a human tPA sequence. The probes were used to.
Southern blot. The tables below summarize the in for mation given in Chapter 2 of this guide. For a. For calculation of other hybridization. The optimum hybridization temperature Thyb for a probe-target hybrid can be calculated. Please note that the for mula you use depends on the hybridization.
If you are using this hybridization buffer Always use this calculation for T hyb. Always use these equations to calculate the optimal hybridization temperature Thyb for. Use these equations to calculate the optimal hybridization temperature Thyb for. The addition. For sequences. Increase [ for A ] or decrease [ for B ] the amount of time you expose the blot to X-ray. Spread the chemiluminescent substrate uni for mly over the surface of the membrane,.
Be for e exposing bag to X-ray fi lm, fl atten any wrinkles between blot and membrane by. Use positively charged Nylon Membranes from Roche Applied. Do not let the membrane dry even slightly be for e adding the chemiluminescent substrate. Reisolate target nucleic acid and check the sample for degradation be for e using it in.
Be very careful when handling the blot during a DIG procedure. Handle it only by the. Do not touch the experimental portion of the. Always start with a fresh blot when per for ming a DIG procedure for the fi rst time; do. This section illustrates how you can evaluate DIG -labeled probes to ensure good results. From the result of Step 1, we chose the probe probe 2 with the greatest sensitivity. We also tried fi ltering the probe through cellulose acetate to try to reduce the observed.
The blot shows good results. The optimization. Figure A. DIG -labeled probes were. Result and conclusions: Probe 2 has the best sensitivity 0. Probes 1 and 3 have a sensitivity of 0. Probe 4 had a sensitivity of only 1 pg. There for e, probes 1 — 3 all. Probe 4 did not have suffi cient sensitivity. Figure B. DIG -labeled probe 2 from Figure. A was used as probe in a series of standard hybridization reactions.
This serves as a test for non-specifi c background binding of the probe. In each case, stringent hybridization. The upper series of photos shows the results when the different probe. The lower series of photos were obtained when the. Result and conclusions: The acceptable range of concentrations for the probe is very narrow.
Both the low left. In contrast, the high. The difference between a good background and a. This background is clearly due solely to. A comparison of the upper and lower series of panels shows that fi ltration lowered the background even further. The unfi ltered probe upper series of photos produces a lot of spotty background. Some of the contamination could be removed from the. Figure C. Filter ed, DIG -labeled probe 2 was used to detect a single-copy gene on a Southern blot.
The target DNA. In each case, samples containing. Weight Marker was included between each set of samples. Following the optimization guidelines determined. Our standard protocol was used for hybridization and chemiluminescent detection. Background is very low. There for e,. Human blood DNA was the chosen target of the customer who. The table below describes ways to get the best results at each stage of the DIG labeling and. For suggestions on improving results of specifi c DIG labeling and detection procedures,.
Autoclave DIG System solutions. Add Tween 20 to solutions after sterilization. Use clean incubation trays. Rigorously clean and rinse trays be for e each use. For Northern blots, use sterilized glass trays for all washing and detection steps.
Labeled vector. If large amounts of labeling by-products are for med e. Thus, for each type of labeling reaction, there is an optimal. Use the digested product directly for hybridization. Please separate multiple entries using comma. You will receive the Roche Newsletter for our new and exciting products and special offers. You will hear about upcoming events, and download the latest technical and scientific literature. Your privacy is ensured. This website uses cookies to provide you with a more responsive and personalized service.
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For life science research only. Not for use in diagnostic procedures. Recombinant Vv-AMP1 displayed non-morphogenic antifungal activity against a broad spectrum of fungi, probably altering the membrane permeability of the fungal pathogens. The expression of this peptide is highly regulated in Vitis vinifera , hinting at an important defense role during berry-ripening.
Plants are constantly subjected to microbial attack, especially phytopathogenic fungi and use various defense strategies to protect themselves against disease. These defenses include the strengthening of the physical cell wall barriers [ 1 ] and the production of chemical and proteinaceous antimicrobial compounds [ 2 - 6 ]. Over the last 15 years it has become evident that small, basic, cysteine-rich peptides also form part of the overall defense of plants against phytopathogens, contributing significantly to the innate immunity of plants [ 7 - 10 ].
It has been suggested that all plants possess such a peptide defense system [ 8 ]. The peptides range from 2—9 kDa in size and the best characterized examples are the thionins and defensins [ 7 , 8 , 10 - 15 ]. Plant defensins are a family of basic, cysteine-rich peptides of between 45—54 amino acids in size.
Although plant defensins are structurally conserved, their overall homology at the amino acid level is low. However, most plant defensins contain eight cysteine residues linked by four disulfide bridges, an aromatic residue at position 11, two glycines at positions 13 and 34 and a glutamate at position 29 numbering according to Rs-AFP1 [ 23 ].
Most plant defensins exhibit some antimicrobial activity, inhibiting the growth of fungi, oomycetes and gram positive bacteria in vitro.
The exact mechanisms underlying the antifungal activity exerted by plant defensins are not known, but there is evidence that plant defensins bind to a specific receptor in the fungal membrane, being sphingolipids, rather than random binding and integration into the phospholipid bilayer of the fungal membranes [ 24 - 31 ].
The majority of defensins have been isolated from plant seeds [ 35 - 43 ], but defensins have also been isolated from leaves [ 23 , 44 ], flowers [ 45 - 49 ], tubers [ 50 ], seedpods [ 38 ], as well as from fruits [ 51 - 53 ]. Although plant defensins play an important role in the preformed defense, some members of the defensin family are also upregulated upon pathogen attack or by environmental stimuli, while the expression of others are strictly developmentally regulated [ 34 , 51 , 52 , 54 - 59 ].
Here we report the isolation and characterization of the first plant defensin from Vitis vinifera. The peptide encoding gene shows a strict tissue-specific and developmentally regulated expression pattern. The peptide is strongly antifungal without inducing morphological changes to the hyphae of the tested fungi, but with an indication of a compromising effect on the fungal membrane.
The ripening-specific expression pattern in berries and the strong in vitro antifungal characteristics of the isolated peptide draws interest to its possible in vivo role in berry defence systems.
Subsequent analyses of the available databases after the completion of the grapevine genome sequence, has yielded other putative defensin sequences results not shown. The sequence was termed Vitis vinifera antimicrobial peptide 1 Vv-AMP1 , because of its homology to the family of plant defensins. Gene structure of Vv-AMP1. The amino acids in yellow represent the signal peptide while red amino acids indicate the mature peptide. Yellow blocks represent the sequence encoding for the signal peptide of Vv-AMP1 and the red block the sequence encoding for the mature Vv-AMP1 peptide.
The intron is indicated as a grey block. Numbering inside each block corresponds to the number of base pairs in each section. Alignment analysis of these two genomic sequences showed that the nucleotide areas upstream and downstream of the Vv-AMP1 open reading were similar, suggesting that a single copy of Vv-AMP1 is present in the V.
Vitis vinifera cv. Each signal indicates a single copy of Vv-AMP1. Vv-AMP1 from V. The deduced amino acid sequences from V. Alignment analysis of the deduced amino acid sequences of the Vv-AMP1 genes isolated from different Vitis species. The major differences are indicated in green, note the additional amino acid at position 15 of the sequences isolated from V.
Vv-AMP1 also displays the following conserved amino acid residues: an aromatic residue at position 11, two glycine residues at positions 13 and 34 and a glutamate at position 29, as well as the eight cysteine residues at positions 4, 15, 21, 25, 36, 46, 48, 52 present in all plant defensins numbering according to Rs-AFP1 [ 23 ]. The consensus sequence common to all defensins is indicated below with numbering according to Rs-AFP1 [ 23 ]. The eight cysteines are indicated in yellow and the aromatic residue at position 11 in green.
The conserved glycines are indicated in blue and glutamate at position 29 in grey. The gap was introduced to have number agreement with Rs-AFP1. The disulfide bridge organization within the Vv-AMP1 sequence is indicated below the consensus sequence. The structure is stabilized by intramolecular disulfide linkages between the eight cysteine residues.
Localization of GFP observed in the apoplasts of the following tissues, organs and cells in the leaf petiole: A cortex; B the guard cells of the stomata and C the trichoma. A1, B1 and C1 show light microscopic photos of these various tissues and organs from the untransformed control. A2, B2 and C2 show the auto-fluorescence from these same fields in the untransformed controls, whereas A3, B3 and C3 indicate GFP expression in the apoplastic regions of these structures in the GFP overexpressing lines.
Northern blot analysis on berries in different stages of development and ripening confirmed that the gene is developmentally regulated. Expression of Vv-AMP1 remained high throughout the rest of the berry ripening stages. Biotic and abiotic induction studies were conducted on Pinotage leaf tissue. Time points indicate the time of tissue collection after the initiation of each induction experiment. The Vv-AMP1 signal hybridized at a molecular weight of bp and the internal standard at bp.
The recombinant fusion protein had a size of 31 kDa, consistent with the predicted size. Recombinant peptide was successfully separated from the cleaved tag, using ion exchange chromatography, and desalted on a C8 column.
Mass spectrometry revealed that the recombinant peptide had a size of 5. Recombinant Vv-AMP1 was tested against several plant pathogenic fungi using a dose-response growth inhibition assay. The activity of Vv-AMP1 on fungal hyphae was assessed by incubating fungal spores in the presence of various concentration of Vv-AMP1 over a 72 hour period, with the IC 50 value being determined after 48 hours of incubation Figure 9A—D. Vv-AMP1 had a severe effect on the accumulation of fungal biomass over time in all of the fungal isolates tested and was most active against F.
Vv-AMP1 was however less effective against F. The necrotrophic fungi B. Treatment of B. The peptide showed no inhibition of A. Antifungal activity of Vv-AMP1 on a panel of plant pathogenic fungi. Microspectrophotometric readings were recorded every 24 hours and compared to the untreated fungal controls. The data is represented as a percentage of fungal growth as compared to the untreated control reactions with no peptide.
Growth inhibition was determined after 48 hours of growth for the Fusarium and B. Microscopical analyses of fungal hyphae treated with Vv-AMP1 showed no signs of the characteristic hyperbranching effect associated with some plant defensins. Vv-AMP1 did, however, severely alter the ability of fungal hyphae to elongate and most hyphal tips had a swollen appearance. Granulation of the hyphal cytoplasm was also observed in most fungi treated with Vv-AMP1 data not shown.
This is an indication that the fungal membranes were compromised by the presence of the Vv-AMP1 peptide. Fluorescent microscope analysis of Propidium Iodide uptake during the membrane permeabilization assay. Afterwards fungal hyphae were stained with Propidium iodide for 10 min, washed with 1XPBS and subjected to fluorescent microscopic analysis.
Vv-AMP1 was tested for its stability at different temperatures using an antifungal growth assay against Botrytis cinerea Figure 11A. Stability assessment of Vv-AMP1. Vv-AMP1 was very sensitive to proteinase K treatment, confirming its proteinaceous nature.
Plant defensins are small, cysteine-rich peptides with a basic nature that exhibit a broad spectrum of antimicrobial activity and have been implicated in the innate defense system of plants. Here we report the isolation and characterization of the first defensin peptide and its encoding gene from Vitis vinifera , the world's most important fruit crop. The bp open reading frame isolated from V. Analysis of the grapevine genome revealed that only one copy of Vv-AMP1 is present in the Vitis vinifera genome and the two hybridization signals observed in the Southern blot analysis are due to heterogeneity in the Vv-AMP1 locus.
Most of the non-vinifera Vitis spp. Bioinformatical analyses on these sequences in comparison with the V. The recently completed grapevine genome sequence revealed additional putative defensin sequences in Vitis vinifera results not shown that could be targeted for isolation and characterization as well.
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