Amersham cdp star detection reagent, by GE Healthcare - Product details

1

Genomic DNA Extraction and Transgene Detection in Plants

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Genomic DNA was extracted from young leaves of greenhouse-grown plantlets using cetyltrimethylammonium bromide method¹⁷. The presence of each of the transferred genes was confirmed by PCR with gene-specific primers (Supplementary Table 2).
For Southern blot, 30 μg of plant genomic DNA was digested overnight at 37°C by 100U of EcoRV, a restriction enzyme that cuts T-DNA constructs used in this study at a single position inside nnHispS coding region. After gel electrophoresis, digestion products were transferred onto Amersham Hybond-N+ membrane (GE Healthcare, UK) and immobilized. The DNA probe was constructed by PCR using cloned synthetic nnluz gene as the template and nnluz-specific primers listed in Supplementary Table 2. Probe DNA was labeled with alkaline phosphatase using the AlkPhos Direct Labeling Kit (GE Healthcare, UK). Prehybridization, hybridization (overnight at 60°C) with alkaline phosphatase-labeled probe, and subsequent washings of the membrane were carried out according to the AlkPhos Direct Labeling Kit protocol. Detection was performed using Amersham CDP-Star detection reagent following the manufacturer’s protocol (GE Healthcare, UK). The signal from the membrane was accumulated on X-ray film (XBE blue sensitive, Retina, USA) in film cassette at room temperature for 24 hours. X-ray films were scanned on Amersham imager 600 (GE Healthcare Life Sciences, Japan).

Mitiouchkina T., Mishin A.S., Somermeyer L.G., Markina N.M., Chepurnyh T.V., Guglya E.B., Karataeva T.A., Palkina K.A., Shakhova E.S., Fakhranurova L.I., Chekova S.V., Tsarkova A.S., Golubev Y.V., Negrebetsky V.V., Dolgushin S.A., Shalaev P.V., Shlykov D., Melnik O.A., Shipunova V.O., Deyev S.M., Bubyrev A.I., Pushin A.S., Choob V.V., Dolgov S.V., Kondrashov F.A., Yampolsky I.V, & Sarkisyan K.S. (2020). Plants with genetically encoded autoluminescence. Nature biotechnology, 38(8), 944-946.

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2

Southern Blot and SDS-PAGE Analysis

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In the Southern blot analysis, genomic DNA was isolated from adults using the method described by Sambrook and Russell [57 ]. Genomic DNA was digested with either BamHI or BglII. Digested genomic DNAs were then separated on a 1.0% agarose gel and subsequently transferred to a Hybond-N+ membrane (GE Healthcare UK Ltd., Buckinghamshire, UK). Southern blot analysis was performed using probes labeled with Amersham AlkPhos Direct Labeling Reagents, and DNA bands were visualized using Amersham CDP-Star Detection Reagent following the manufacturer’s guidelines (GE Healthcare). SDS-PAGE analysis of hemolymph was performed according to the method of Mine et al. [30 (link)]. Hemolymph (1 μL) was treated with SDS sample buffer for 2 min at 95°C. Samples were separated by electrophoresis on 10% SDS-polyacrylamide gel and stained with Quick-CBB PLUS (Wako, Osaka, Japan).

Sakai H., Sumitani M., Chikami Y., Yahata K., Uchino K., Kiuchi T., Katsuma S., Aoki F., Sezutsu H, & Suzuki M.G. (2016). Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori. PLoS Genetics, 12(8), e1006203.

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3

Tobacco DNA Blot Analysis

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Tobacco genomic DNA (20 μg) was digested overnight at 37 °C with 60 U EcoRI which cut the T-DNA of p2120 and p455 at a single position (see Producing genetic constructs for Agrobacterium-mediated plant transformation). The fragments were separated on a 0.9 % agarose gel and transferred to a positive-charged nylon membrane Hybond N+ (GE Healthcare,UK) by capillary blotting following the manufacturer’s instructions. The DNA probe was constructed by PCR using plasmid p2120 as the template, and primers gus-1 and gus-2 (Additional file 1: Table S3). Probe DNA (740 bp) was labeled with alkaline phosphatase using Amersham Gene Image AlkPhos Direct Labelling and Detection System (GE Healthcare, UK). Prehybridization, hybridization (overnight at 60 °C) with alkaline phosphatase-labeled probe, and subsequent washings of the membrane were carried out according to the AlkPhos Direct Labeling System protocol. Detection was performed using CDP-Star detection reagent following the manufacturer’s directions (Amersham CDP-Star Detection reagent, GE Healthcare, UK).

Komakhin R.A., Vysotskii D.A., Shukurov R.R., Voblikova V.D., Komakhina V.V., Strelnikova S.R., Vetchinkina E.M, & Babakov A.V. (2016). Novel strong promoter of antimicrobial peptides gene pro-SmAMP2 from chickweed (Stellaria media). BMC Biotechnology, 16, 43.

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4

Plum RNA Extraction and Northern Blot

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An extracted plum total RNA (20 μg) was loaded onto 15% polyacrylamide gel containing 7 M urea, and then electrotransferred onto a membrane (Hybond-N+, GE Healthcare, United Kingdom). siRNA bands were probed within the PPV CP 897 bp PCR fragment that was labeled with alkaline phosphatase using the Amersham Gene Image AlkPhos Direct Labeling and Detection System (GE Healthcare, United Kingdom). Detection was performed using CDP-Star detection reagent following manufacturer’s directions (Amersham CDP-Star Detection reagent, GE Healthcare, United Kingdom).

Sidorova T., Mikhailov R., Pushin A., Miroshnichenko D, & Dolgov S. (2019). Agrobacterium-Mediated Transformation of Russian Commercial Plum cv. “Startovaya” (Prunus domestica L.) With Virus-Derived Hairpin RNA Construct Confers Durable Resistance to PPV Infection in Mature Plants. Frontiers in Plant Science, 10, 286.

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5

Extraction and Detection of siRNA

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Total siRNA was extracted from young leaves (1 g) using an acid guanidinium thiocyanate–phenol–chloroform extraction method as described in [58 (link),59 (link)]. An extracted rootstock 146-2 total RNA (20 μg) was loaded onto 15% polyacrylamide gel containing 7 M urea, and then (after electrophoretic separation in PAAG) electrotransferred onto a membrane (Hybond-N+, GE Healthcare, Amersham Bioscience, Amersham, UK). siRNA bands were probed within the eIF(iso)4G or eIF(iso)4E 0.6kbp PCR fragments that were labeled with alkaline phosphatase using the Amersham Gene Image AlkPhos Direct Labeling and Detection System (GE Healthcare, Amersham Bioscience, Amersham, UK). Prehybridization, hybridization (incubated at 42 °C overnight) with alkaline phosphatase labeled probe, and subsequently washings of the membrane were carried out according to the AlkPhos Direct Labeling Kit protocol. Detection was performed using CDP-Star detection reagent following the manufacturer’s directions (Amersham CDP-Star Detection reagent, GE Healthcare, Amersham, UK).

Mourenets L., Pushin A., Timerbaev V., Khmelnitskaya T., Gribkov E., Andreev N, & Dolgov S. (2022). Effect of Gene Silencing of Translation Initiation Factors eIF(iso)4G and eIF(iso)4E on Sour Cherry Rootstock Resistance to Sharka Disease. International Journal of Molecular Sciences, 24(1), 360.

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6

Genomic DNA Extraction and Transgene Detection in Plants

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Genomic DNA was extracted from young leaves of greenhouse-grown plantlets using cetyltrimethylammonium bromide method¹⁷. The presence of each of the transferred genes was confirmed by PCR with gene-specific primers (Supplementary Table 2).
For Southern blot, 30 μg of plant genomic DNA was digested overnight at 37°C by 100U of EcoRV, a restriction enzyme that cuts T-DNA constructs used in this study at a single position inside nnHispS coding region. After gel electrophoresis, digestion products were transferred onto Amersham Hybond-N+ membrane (GE Healthcare, UK) and immobilized. The DNA probe was constructed by PCR using cloned synthetic nnluz gene as the template and nnluz-specific primers listed in Supplementary Table 2. Probe DNA was labeled with alkaline phosphatase using the AlkPhos Direct Labeling Kit (GE Healthcare, UK). Prehybridization, hybridization (overnight at 60°C) with alkaline phosphatase-labeled probe, and subsequent washings of the membrane were carried out according to the AlkPhos Direct Labeling Kit protocol. Detection was performed using Amersham CDP-Star detection reagent following the manufacturer’s protocol (GE Healthcare, UK). The signal from the membrane was accumulated on X-ray film (XBE blue sensitive, Retina, USA) in film cassette at room temperature for 24 hours. X-ray films were scanned on Amersham imager 600 (GE Healthcare Life Sciences, Japan).

Mitiouchkina T., Mishin A.S., Somermeyer L.G., Markina N.M., Chepurnyh T.V., Guglya E.B., Karataeva T.A., Palkina K.A., Shakhova E.S., Fakhranurova L.I., Chekova S.V., Tsarkova A.S., Golubev Y.V., Negrebetsky V.V., Dolgushin S.A., Shalaev P.V., Shlykov D., Melnik O.A., Shipunova V.O., Deyev S.M., Bubyrev A.I., Pushin A.S., Choob V.V., Dolgov S.V., Kondrashov F.A., Yampolsky I.V, & Sarkisyan K.S. (2020). Plants with genetically encoded autoluminescence. Nature biotechnology, 38(8), 944-946.

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7

Validation of Genomic Integration by Southern Blotting

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Proper integration of the pSN054-1437000-3xHA vector was confirmed by Southern blotting. Genomic DNA from the parent and edited lines was isolated (QIAamp DNA Blood Miniprep Kit), and 10 μg of each was digested with HinDIII, separated overnight on a 0.7% agarose gel and transferred to nylon (Nytran SuPerCharge TurboBlotter, 0.45 μm, GE Healthcare) overnight. The blot was then probed with the left homologous region (PCR product of 14APT-1/14APT-2) labelled with alkaline phosphatase (Amersham AlkPhos Direct Labeling Kit; GE Healthcare) in hybridization buffer (Amersham) at 55°C overnight, washed twice each in primary wash buffer (120 g/l urea, 1 g/l SDS, 100 ml/l 0.5 M sodium phosphate pH 7, 8.7 g/l NaCl, 2 g/l Amersham blocking reagent) and secondary wash buffer (6.05 g/l Tris base, 5.6 g/l NaCl, 2 ml/l 1 M MgCl₂, pH 10), then detected with Amersham CDP-Star Detection Reagent (GE Healthcare) and exposed to blue autoradiography film (MidSci, BX810) overnight.
Additional validation by western blotting was done exactly as described in Polino et al. (2020) (link) (section ‘Validation of PMV^APT line’).

Polino A.J., Hasan M.M., Floyd K., Avila-Cruz Y., Yang Y, & Goldberg D.E. (2023). An essential endoplasmic reticulum-resident N-acetyltransferase ortholog in Plasmodium falciparum. Journal of Cell Science, 136(6), jcs260551.

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8

Genomic DNA Extraction and Southern Blot Analysis

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Genomic DNA was extracted from 1 g of young leaf tissue as described by Ince et al. [42 ]. NsiI- or BspHI and EcoRV-digested genomic DNA (50 μg) was resolved on a 0.8 % agarose gel and blotted by capillary transfer onto Hybond N+ membrane (GE Healthcare). For dot blots, 100 ng of plasmid DNA was pipetted and UV crosslinked to the membrane. A PCR product amplified with ANT1-specific primers TC080F and C2R or LIR primers TC101F and TC246R (Table S1 in Additional file 2) was used as a probe. Purified PCR product (200 ng) was labeled using the Amersham AlkPhos Direct Labeling and Detection System (GE Healthcare) and hybridized to membranes at 60 °C overnight. Membranes were processed according to the manufacturer’s recommendations. Probes were detected using the Amersham CDP-Star Detection Reagent (GE Healthcare), and signals were captured on X-ray film (Amersham Hyperfilm ECL, GE Healthcare). For re-probing, membranes were stripped in 0.5 % SDS solution at 60 °C.

Čermák T., Baltes N.J., Čegan R., Zhang Y, & Voytas D.F. (2015). High-frequency, precise modification of the tomato genome. Genome Biology, 16, 232.

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9

RNA Extraction and Northern/Dot Blot Analysis

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Fresh mycelia cultured for 3 days were used to extract the total RNA. The probe is a 747-bp segment amplified with pMD18-P1 as a template and the OMFA/OMR2 as primers. The northern blot was carried out following the manual of alkaline Direct Labeling System and Amersham Hybond-N+ (GE Healthcare, Little Chalfont, United Kingdom). For northern blot, total RNA was denatured for 10 min at 70°C and separated using denatured agarose gel with formaldehyde and then transferred to a positively charged nylon membrane (GE) using alkaline transfer solution (0.01 M NaOH, 3 M NaCl). For the dot blot, RNA samples (2 μ μl) were added on the positively charged nylon membrane and dried out. The dried nylon membrane was cross-linked using a UV cross-linker. The hybridization was performed overnight in a glass tube at 55°C. The chemiluminescent signal generation and detection were performed using an Amersham CDP-Star Detection Reagent (GE).

Wang Q., Mu F., Xie J., Cheng J., Fu Y, & Jiang D. (2020). A Single ssRNA Segment Encoding RdRp Is Sufficient for Replication, Infection, and Transmission of Ourmia-Like Virus in Fungi. Frontiers in Microbiology, 11, 379.

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10

Southern Blot and Mutagen Sensitivity Assay

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Southern blot hybridization was carried out as previously described (Ando et al., 2009b) . Genomic DNA (10 μg) was digested with SacI and XhoI, size-fractioned by electrophoresis (1% agarose), and transferred to Amersham Hybond-N + membrane (GE Healthcare Ltd., Buckinghamshire, UK). Probes used for hybridization correspond to a 0.8 kb region of the 5'-fragment of lig4 gene and a 0.6 kb region of the ura5 gene, and were generated using Lig4 F2 and Lig4 R1 ApaI, and ura5 start F and ura5 stop R primer sets (Table S1), respectively. Southern hybridization was performed with Amersham Alkphos Direct Labeling Reagents (GE Healthcare) and Amersham CDP-Star Detection Reagent (GE Healthcare), and chemiluminescence was detected by ImageQuant LAS 4000 mini system (GE Healthcare).
2.9 Mutagen sensitivity of Δlig4::ura5 strain Sensitivity to chemical mutagens was evaluated by the spot test (Kato et al., 2004; Mizutani et al., 2008) . Methyl methanesulfonate (MMS), at 0, 0.01, 0.02, 0.03, 0.04, and 0.005% [w/v], was added to SC agar medium supplemented with 0.05 mg/ml uracil.
Spores were spotted and grown on the agar plates for 8 days.

Kikukawa H., Sakuradani E., Ando A., Okuda T., Ochiai M., Shimizu S, & Ogawa J. (2015). Disruption of lig4 improves gene targeting efficiency in the oleaginous fungus Mortierella alpina 1S-4. Journal of biotechnology, 208.

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Amersham cdp star detection reagent

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12 protocols using amersham cdp star detection reagent

Genomic DNA Extraction and Transgene Detection in Plants

Southern Blot and SDS-PAGE Analysis

Tobacco DNA Blot Analysis

Plum RNA Extraction and Northern Blot

Extraction and Detection of siRNA

Genomic DNA Extraction and Transgene Detection in Plants

Validation of Genomic Integration by Southern Blotting

Genomic DNA Extraction and Southern Blot Analysis

RNA Extraction and Northern/Dot Blot Analysis

Southern Blot and Mutagen Sensitivity Assay

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