Monday, November 10, 2003 Description Isogen RNA Extraction Protocol Procedure This protocol is for 200mg of leaf tissue Add 0.25ml of ddH2O and 0.75ml of Isogen-LS to eppendorf tube (one per sample) Vortex to mix Hold mortar in liquid N2...
Friday, November 07, 2003 Description Methods for Plant RNA Isolation Procedure AMES/Chloroform extraction in our lab to extract total RNA from potato leaves for viroid analysis. The protocol is as follows: AMES Buffer (for 200mL):11.7g Na...
Description With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Procedure A. Permeabilization of...
Tuesday, October 21, 2003 Description RNase Protection and Immunoprecipitation of Nuclear Extracts Procedure A. Prepare Beads (for 15 reactions) 1. Resuspend 60 mg of Protein A-Sepharose CL-4B or 375 l Protein G-Sepharose in 7.5 ml of NET2...
Tuesday, October 21, 2003 Description RNase T1 is an endonuclease that specifically cleaves 3' of G residues in RNA. This protocol uses RNase T1 for RNA mapping studies. Procedure 1. Dry the RNA sample in either a vacuum centrifuge or a ly...
Description This protocol can be used to quantitate an mRNA of interest if the RNA probe is present in molar excess over the target mRNA. A labeled RNA transcript is hybridized to an mRNA population and any non-hybridized (single stranded)...
Monday, October 20, 2003 Description RNA Primer Extension Procedure 1. Precipitate the RNA with 5' end-labeled primer in 0.3 M Sodium Acetate with 2.5 volumes of 100% Ethanol (See Hint #1). 2. Mix well by inversion, microcentrifuge to pell...
Monday, October 20, 2003 Description Reverse Transcriptase Assay Using DEAE-Cellulose Procedure 1. Take 10 l of medium from the wells of a 96-well microtiter plate containing a confluent cell culture of the desired cell line and put it int...
Monday, October 20, 2003 Description This protocol describes the desilylation or 2' deprotection of synthetic RNA. Procedure 1. After synthesis on the 1 M scale, take the column apart with pliers and deposit the resin in a glass deprotecti...
Monday, October 20, 2003 Description RNase Protection and Immunoprecipitation of Nuclear Extracts Procedure A. Prepare Beads (for 15 reactions) 1. Resuspend 60 mg of Protein A-Sepharose CL-4B or 375 l Protein G-Sepharose in 7.5 ml of NET2...