Amersham hybond xl

Manufactured by Cytiva

Amersham Hybond-XL is a nylon-based membrane designed for the transfer and immobilization of nucleic acids, proteins, and other biomolecules during blotting procedures. It provides high binding capacity and optimal transfer efficiency.

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Lab products found in correlation

1 kb plus dna ladder, by Thermo Fisher Scientific (2 mentions) Amersham rapid hybridization buffer, by Cytiva (2 mentions) Typhoon fla 9500, by GE Healthcare (2 mentions) Quick dna fungal bacterial miniprep kit, by Zymo Research (2 mentions) Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) T4 polynucleotide kinase, by New England Biolabs (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) G 32 p atp, by PerkinElmer (1 mentions)

Close spelling variants of this product

Amersham Hybond Hybond-XL

3 protocols using amersham hybond xl

m6A RNA Enrichment and Detection

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PolyA+ RNA selection was performed twice using Dynabeads mRNA DIRECT Purification Kit (Invitrogen, Cat. #61011) as described in the user manual. 2x polyA+ RNA samples were spotted onto the membrane, Amersham Hybond-XL (Cytiva, Cat# RPN303s). The membrane was completely dried and crosslinked in a UV STRATALINKER 1800 using the automatic function. The membrane was then blocked for 10 min using sterile RNase, DNase-free TBST + 5% skim milk. The m⁶A primary antibody was then added at a concentration of 1:1,000 in TBST + 5% skim milk at 4°C, overnight. The membrane was washed four times in TBST and then incubated with the secondary anti-rabbit antibody (1:5,000) for 1 h in TBST + 5% skim milk. The membrane was washed four times in TBST and exposed on the ChemiDoc imaging system (Bio-Rad) using Pierce ECL Western Blotting substrate.

Kim K.L., van Galen P., Hovestadt V., Rahme G.J., Andreishcheva E.N., Shinde A., Gaskell E., Jones D.R., Shema E, & Bernstein B.E. (2021). Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution. Cell Reports Methods, 1(5), 100061.

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Targeted Gene Deletion in P. aeruginosa

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Genomic DNA was isolated from WT and G2L4 knock-out P. aeruginosa AZPAE12409 by using a Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research) according to the manufacturer’s protocol. The DNA was digested with PstI and EcoRI and run in a 1% agarose gel alongside a 1-kb Plus DNA Ladder (Invitrogen) that was 5’-labeled with [γ–³²P]-ATP (6000 Ci/mmol; Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs). After electrophoresis, the gel was blotted onto an Amersham Hybond-XL (Cytiva) membrane by overnight capillary transfer. The membrane was washed 3 times with 25 mL 6X SSC, dried, and UV irradiated to cross-link the DNA to the membrane (120 mJ; Stratalinker UV Crosslinker 2400). Hybridization was done with a 5’-labeled targetron probe (200 bp PCR product obtained using G2L4 RT targetron probe primers; Table S4) in a hybridization tube with Amersham Rapid-hybridization Buffer (Cytiva) for 2.5 h at 60°C. After washing twice with 2X SSC plus 0.1 % SDS, the membrane was dried and scanned with a phosphorimager (Typhoon FLA 9500; GE Healthcare).

Park S.K., Mohr G., Yao J., Russell R, & Lambowitz A.M. (2022). Group II Intron-like Reverse Transcriptases Function in Double-Strand Break Repair. Cell, 185(20), 3671-3688.e23.

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Profiling Genomic DNA in P. aeruginosa

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Genomic DNA was isolated from WT and G2L4 knock-out P. aeruginosa AZPAE12409 by using a Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research) according to the manufacturer's protocol. The DNA was digested with PstI and EcoRI and run in a 1% agarose gel alongside a 1-kb Plus DNA Ladder (Invitrogen) that was 5'-labeled with [g-32 P]-ATP (6000 Ci/mmol; Perkin Elmer) usingT4 polynucleotide kinase (New England Biolabs). After electrophoresis, the gel was blotted onto an Amersham Hybond-XL (Cytiva) membrane by overnight capillary transfer. The membrane was washed 3 times with 25 mL of 6x SSC, dried, and UV irradiated to cross-link the DNA to the membrane (120 mJ; Stratalinker UV Crosslinker 2400). Hybridization was done with a 5'-labeled targetron probe (200 bp PCR product obtained using G2L4 RT targetron probe primers; Table S2) in a hybridization tube with Amersham Rapid-hybridization Buffer (Cytiva) for 2.5 h at 60˚C. After washing twice with 2x SSC plus 0.1 % SDS, the membrane was dried and scanned with a phosphorimager (Typhoon FLA 9500; GE Healthcare).

Park S.K., Mohr G., Yao J., Russell R., & Lambowitz A.M. (2022). Group II Intron-Like Reverse Transcriptases Function in Double-Strand Break Repair by Microhomology-Mediated End Joining.

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