Northern blotting for miRNA detection was performed with minor modifications as described previously [4 (link)]. Briefly, 10 μg of RNA denatured in Ambion gel loading buffer II (Life technologies) at 75°C for 15 min was loaded and ran on a 15% TBE-Urea gels (Invitrogen, Carlsbad, CA, USA) at 180 V for 1 h. The gel was then transferred onto a nylon membrane using an iBLOT DNA transfer stack (Invitrogen) as per the manufacturer's instructions. Subsequently, the membrane was crosslinked at 1200 kJ using a STRATALINKER (Stratagene, La Jolla, CA, USA). The membrane was then prehybridized in the prehyb buffer (Sigma-Aldrich) at 37°C for 1 h followed by the addition of 1.2 pmol/mL of LNA-modified 5′ and 3′ DIG-labeled hsa-miR-1290 probe (Exiqon) to the buffer and hybridization overnight at 37°C. The next day, the membrane was washed for 5 min with a low stringency wash buffer (2 × SSC, 0.1% SDS) followed by 2 × washes for 20 min each with a high stringency wash buffer (0.5 × SSC, 0.1% SDS) and a final wash for 20 min with ultrahigh stringency wash buffer (0.1 × SSC, 0.1% SDS). After the washes, the membrane was blocked for 1 h with 1 × blocking buffer for 1 h followed by incubation with anti-DIG AP antibody in a 1: 20 000 dilution in blocking buffer. Finally, DIG signal development was carried out using the DIG wash and block buffer set.
Iblot dna transfer stack
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBLOT DNA transfer stack is a laboratory equipment used for the transfer of DNA samples from an electrophoresis gel to a membrane for further analysis. It facilitates the efficient and reliable transfer of DNA fragments, enabling researchers to perform downstream applications such as Southern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Prehyb buffer, by Merck Group (2 mentions)
Ambion gel loading buffer 2, by Thermo Fisher Scientific (2 mentions)
Tbe urea gel, by Thermo Fisher Scientific (2 mentions)
Lna modified 5 and 3 dig labeled hsa mir 1290 probe, by Qiagen (2 mentions)
Dig easy hyb, by Roche (2 mentions)
Pcr dig probe synthesis kit, by Roche (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Cdp star ready to use, by Roche (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Dig dna labeling and detection kit, by Roche (1 mentions)
Amersham typhoon biomolecular imager, by GE Healthcare (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Dig dna labelling and detection kit, by Roche (1 mentions)
Dig wash and block buffer set, by Roche (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
5 protocols using iblot dna transfer stack
1
Northern Blotting for miRNA Detection
2
Southern Blot Analysis of Genomic DNA
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For Southern blot analysis, plasmid, bacmid and viral genomic DNA (1 µg) was digested overnight with NsiI and XbaI. The digested DNA was loaded on a 2% agarose gel and electrophoresis was performed for 8 hours at 25 V. Using the iBlot Dry Blotting System (Invitrogen), the DNA was then transferred to the iBlot DNA Transfer Stack (Invitrogen) containing a positively charged nylon membrane. The membrane was incubated in 1.5 mol/l NaCl/0.5 mol/l NaOH denaturing solution for 10 minutes immediately after transfer and air-dried. After UV cross-linking at 130 mJ/cm2, the membrane was hybridized overnight with DIG Easy Hyb (Roche, Indianapolis, IN). DIG-labeled probes targeting the FokI region of the TALEN expression cassette were synthesized using the PCR DIG Probe Synthesis Kit (Roche). Following hybridization, the membrane was stringently washed, blocked, and then incubated with an anti-digoxigenin-AP conjugate (DIG DNA Labeling and Detection Kit, Roche) that was detected by CDP-Star, ready-to-use (Roche). The membrane bearing DNA was exposed to Chemiluminescent Image Analyzer (ImageQuant LAS 4000 mini, GE Healthcare Biosciences, Pittsburgh, PA) for 15 minutes. Primers used for synthesizing DIG-labeled probes are listed in Supplementary Table S1 .
3
tRNA Isolation and Northern Blot Analysis
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tRNAs were isolated following the general protocol A or B, omitting the oxidation by NaIO4. Two to three micrograms of RNA was loaded onto an acidic urea PAGE gel (9% acrylamide (19:1), 100 mM sodium acetate pH 5, 8 M urea) and the gel was run for 12–16 hours, using 100 mM sodium acetate as running buffer, at 6 W constant power. The gel was stained with SYBR Gold (Invitrogen) to identify the tRNAs and an appropriate section of the gel was cut and blotted using iBlot DNA Transfer Stack (Invitrogen) with the iBlot Dry Blotting System. The tRNAs were cross-linked to the membrane (Stratalinker UV Crosslinker 2400), which was subsequently blocked in Ambion ULTRAhyb-Oligo buffer (Invitrogen) for 30 min. The biotinylated DNA probe was added to a final concentration of 0.2 µg ml−1 and hybridized overnight at 37 °C and 160 rpm. The membrane was washed 3 times with 20 ml 0.5x TBE buffer and transferred into 15 ml Odyssey blocking buffer for 20 min before addition of IRDye 800CW Streptavidin (LI-COR) to a final concentration of 0.2 µg ml−1. Finally, the membrane was washed 3 times with 20 ml 0.5x TBE and imaged on an Amersham Typhoon Biomolecular Imager (GE) using the IR long range emission filter.
4
Detecting miRNA Expression by Northern Blotting
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Northern blotting for miRNA detection was performed with minor modifications as described previously.46 (link), 47 Briefly, 10 μg of RNA denatured in Ambion gel loading buffer II (Life technologies) at 75°C for 15 min was loaded and ran on a 15% TBE-Urea gels (Invitrogen, Carlsbad, CA, USA) at 180 V for 1 h. The gel was then transferred onto a nylon membrane using an iBLOT DNA transfer stack (Invitrogen) as per the manufacturer's instructions. Subsequently, the membrane was crosslinked at 1200 kJ using a STRATALINKER (Stratagene, La Jolla, CA, USA). The membrane was then prehybridized in the prehyb buffer (Sigma) at 37°C for 1 h followed by addition of 1.2 pmol/ml of LNA-modified 5′ and 3′ DIG-labeled hsa-miR-1290 probe (Exiqon) to the buffer and hybridization overnight at 37°C. The next day, the membrane was washed for 5 min with a low stringency wash buffer (2 × SSC, 0.1% SDS) followed by 2 × washes for 20 min each with a high stringency wash buffer (0.5 × SSC, 0.1% SDS) and a final wash for 20 min with ultrahigh stringency wash buffer (0.1 × SSC, 0.1% SDS). After the washes, the membrane was blocked for 1 h with 1 × blocking buffer for 1 h followed by incubation with Anti-DIG AP antibody in a 1 : 20 000 dilution in blocking buffer. Finally, DIG signal development was carried out using the DIG wash and block buffer set.
5
Southern Blot Analysis of Genomic DNA
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For Southern blot analysis, 15 μg of genomic DNA was digested with 15 μl of AfeI or NdeI restriction enzymes (New England BioLabs, Ipswich, MA) at 37 °C overnight. As a positive control, 0.5 ng of plasmid DNA was digested with 0.25 μl of AfeI or NdeI restriction enzymes at 37 °C for 2 h. Subsequently, the digested DNA was separated on 1% agarose gel at 20 Volt in cold room overnight. The DNA was then transferred to a nylon membrane provided in the iBlot ® DNA Transfer Stack (Invitrogen) with the pre-set program 8 of the iBlot Gel Transfer Device (Invitrogen) for 7 min. The membrane was denatured with 1.5 M NaCl/0.5 M NaOH solution for 10 min and air-dried for 10 min before cross-linking by ultraviolet light at 120 mJ/cm2 twice. The membrane was then pre-hybridized with DIG Easy Hyb (Roche Diagnostics, Indianapolis, IN) for 1 h and then hybridized overnight at the hybridization temperature of 48 °C with DIG-labelled probe. The probe was synthesized using the PCR DIG Probe Synthesis Kit (Roche Diagnostics). After hybridization, the membrane was washed and blocked using DIG Wash and Block Buffer Set (Roche Diagnostics) according to the manufacturer’s instructions. Lastly, the membrane was incubated with anti-digoxigenin-AP antibody for 1.5 h and developed with CDP-Star provided in the DIG DNA Labelling and Detection Kit (Roche Diagnostics) followed by visualization.
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