MssI (PmeI) (5 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Maj9-2 plasmid containing mouse major satellite repeat DNA (46) was digested with MssI (PmeI) according to Thermo Scientific instructions.

Protocol tips
Maj9-2 plasmid containing mouse major satellite repeat DNA (46) was digested with MssI (PmeI) according to Thermo Scientific instructions.

Publication protocol

Maj9-2 plasmid containing mouse major satellite repeat DNA (46) was digested with MssI (PmeI) according to Thermo Scientific instructions. DIG-labelled MSR transcripts were generated in vitro from linearized Maj9-2 plasmid using SP6/T7 Transcription Kit (Roche) and DIG RNA Labeling Mix (Roche) at 37°C for 3 h. DIG-labelled Control transcripts were generated from a mixture of pSPT18- and pSPT19-neo-DNA (provided by the kit) cleaved with EcoRI. After DNase digestion, DIG-labeled RNAs were purified by acid-phenol chloroform (pH 4.5) extraction (Invitrogen) and isopropanol precipitation. MSR transcript binding and processing assays were performed by incubating 5 μg of DIG-labeled MSR and control transcripts with DICER complexes that were immunoprecipitated from 18 dpp mouse testes as described above in the binding buffer (20 mM Tris–HCl pH 7.4, 50 mM NaCl, 5 mM MgCl2 and 0.3% glycerol) at 35°C for 60 min. For binding assay, beads containing immunoprecipitation complexes were washed and bound RNAs were extracted (TRIsure, Bioline), and either directly applied on nylon membrane (Hybond-N+, Amersham Biosciences, Little Chalfont, UK) for RNA dot blotting or run into a 2.75% denaturing formaldehyde agarose gel in HT buffer (47) and capillary transferred overnight onto a nylon membrane in 10xSSC at +4°C. For processing assay, total RNA was isolated from whole reaction mixture, run into a denaturing 15% polyacrylamide-urea gel in TBE buffer and transferred using a semi-dry transfer system (Trans-Blot Turbo, Bio-Rad) in 0.5× TBE, 20V for 90 min at +4°C. RNA was cross-linked onto the nylon membrane by UVP CL-1000 Ultraviolet Cross linker (400 mJ for 40 s). Membranes were blocked with 4% BSA in Maleic acid buffer (0.1 M Maleic acid, 0.15 M NaCl, pH 7.5) at RT for 60 min. Subsequently, membranes were incubated with the alkaline phosphatase (AP)-conjugated anti-DIG-AP antibody (1:10 000 dilution, 11093274910, Roche) in Maleic acid buffer containing 1% BSA for 30 min, washed with 0.3% Tween 20 in Maleic acid buffer and equilibrated in DIG detection buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5). DIG signal was visualized by incubating with 5–10 drops of a chemiluminescent alkaline phosphatase substrate (CSPD ready-to-use, Roche) for 20 min at RT followed by detection with ImageQuant LAS 4000 Biomolecular Imager (GE Healthcare).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes PmeI / MssI using MssI (PmeI) (5 U/µL) from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for MssI (PmeI) (5 U/µL) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes PmeI / MssI using MssI (PmeI) (5 U/µL) from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.