Rna slot blot protocol

RNA dot blot and slot blot (() Beverly Faulkner-Jones) This methods allows the rapid analysis of numerous small samples for the sequence of interest and is less time consuming than the gel electrophoresis methods. 'Dots' or 'slots' of RNA are made onto a filter using a manifold and the filter is then hybridized with a labeled probe. A dot blot (or slot blot) is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by DUNIAPOKERV.XYZd, the sample is applied directly on a membrane in a single spot, and the blotting procedure is performed. PROTEIN BIO-DOT (‘SLOT BLOT’) Adapted from existing protocols by Vinh Pham. Last modified: December 2, MATERIALS: Cracking buffer stock solution PMSF (mg/ml) Urea 48g Glass beads (umum) SDS 5g 2-Mercaptoethanol 1M Tris-HCl, pH 4ml Nitrocellulose membrane M EDTA 20ul Whatman paper.

RNA Slot Blotting

My bio page Edit user profile Signature Reset the password. Disassemble the blotting manifold and remove the nitrocellulose membrane and put the membrane between 3 M paper. Apply the mixture to QIAamp Mini spin column in a 2 ml collection tube without wetting the rim. Representative data For representative data, please see the paper of Shen et al. It is then incubated with a primary antibody followed by a detection antibody or a primary antibody conjugated to a detection molecule commonly HRP or alkaline phosphatase.

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PolyA RNA Slot Blots

Total RNA should be in approx. Chill on ice. Take nitrocellulose, soak 10 min in water and then 15 min in 4X SSC. Put the wetted nitrocellulose on top of the filter paper. Put the top part of the apparatus on and screw it tightly. Add the sample from Step 1. Let the sample stay in the well for 30 min. Take 0. Put this tube in a 1. Spin 1 min at rpm.

Wash 2x with 0. Jump to: navigation , search. Poly A RNA determination 1. Apply vacuum, suck all the samples in—wells should be visibly dry. Wash twice with 10X SSC. Wet the membrane in 4X SSC for 10 min. Pre-hybridize membrane in the Wash 3x at room temp with 0. Monitor with Geiger counter—membrane should be uniformly hot.

Solutions: Denaturing soln: 5 ml Formamide 1. Use Sephadex G Spin column to remove unincorporated nucleotides spin at rpm. Making the Sephadex G column: Take 0. Category : Protocols. Navigation menu Personal tools Log in. Namespaces Page Discussion. Views Read View source View history. Navigation Main page Recent changes Help. This page was last modified on 9 October , at Privacy policy About Buratowski Lab wiki Disclaimers.

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Ultraviolet UV irradiation induces helix distorting photolesions such as cyclobutane pyrimidine dimers CPD and pyrimidine-pyrimidone photoproducts PP which threaten genomic integrity if unrepaired. Figure 3. Representative data for slot blot repair assay. Control siRNA target cyclophilin B housekeeping gene.

This figure was adapted from Kang et al. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.

You are highly recommended to post your data including images for the troubleshooting. My bio page Edit user profile Signature Reset the password. A peer-reviewed protocol journal. No publication fee; no access fee. Jeong-Min Park. Tae-Hong Kang thkang dau. Original research article A brief version of this protocol appeared in:. Similar Protocols. Reproducibility Feedback. Share your feedback. Abstract Ultraviolet UV irradiation induces helix distorting photolesions such as cyclobutane pyrimidine dimers CPD and pyrimidine-pyrimidone photoproducts PP which threaten genomic integrity if unrepaired.

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Remove the medium, wash with warming 1x PBS once. Note: It is necessary to remove the plastic lid before UV irradiation. Incubate cells in culture medium for repair for the indicated times before harvest them.

Thus for obtaining the optimal time courses for CPD and PP repair a pioneer experiment with various time courses should be performed. The following time courses are good to be tested for uncharacterized cells. Trypsinize and collect each sample in a 1. Cell pellets can be stored at deep freezer up to 1 month for later DNA preparation. Briefly centrifuge to remove drops from the inside of the lid. Apply the mixture to QIAamp Mini spin column in a 2 ml collection tube without wetting the rim.


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