Tuesday, November 18, 2003 Description Appropriate vector DNA is digested with the relevent restriction endonucleases and the 5'-terminal phosphates removed by CIP. The DNA fragment to be cloned is typically prepared by endonuclease digest...
Tuesday, November 18, 2003 Description 3' Rapid Amplification of cDNA Ends (RACE) PCR This technique is used to obtain the 3'end of a cDNA, it requires some sequence information internal to the mRNA under study. The sequence information ob...
Tuesday, November 18, 2003 Description Disruption by Fusion PCR Procedure 1) In separate PCR reactions, amplify the 5' and 3' ends of the gene of interest with primers about 200 bases apart. Primer 2 should begin with 24 nts complementary...
Tuesday, November 18, 2003 Description UV Mutagenesis Procedure 1) Grow cells 2-3 days to stationary phase 2) Wash cells 2x with water 3) Count cells (vortex well to separate cells as much as possible) 4) Resuspend to 2 x 108 cells/ml 5) T...
Tuesday, November 18, 2003 Description Vectorette PCR of Yeast DNA Procedure 1) Cut 1-3 g of clean DNA overnight with 8-10U of blunt cutting enzyme in 20l Most problems come from dirty, uncut DNA. Phenol/glass bead/RNase prepared DNA works...
Tuesday, November 18, 2003 Description Yeast Colony PCR Procedure PCR mixture Combine reaction mix on ice: 2.5 μl 10X Colony PCR Buffer 1.5 μl 25 mM MgCl2 0.5 μl 10 mM dNTP's 10 pmols of each primer 1.25 μl 20% Triton X-100 0.25 μl Ta...
Monday, November 10, 2003 Description Leaf GUS Assay Procedure This protocol is for small samples (usually single leaf from 21DAI plants), scale up for larger samples As there are usually many samples, we use a microtitre plate and plate r...
Monday, November 10, 2003 Description Inverse PCR Protocol This method can be used directly for pBIN19 or pBI101 based T-DNA inserts Procedure Digestion reaction: 2.5g genomic DNA (CTAB) 1x 10x enzyme buffer 20-40U restriction enzyme xl dd...
Sunday, October 19, 2003 Description This protocol describes a competitive ligand binding assay for cortical neurotrophin receptors. Following binding in the presence of competitor, the bound radiolabeled ligand is cross-linked to the rece...
Sunday, October 19, 2003 Description Gel Mobility Shift Assay Using Magnesium Chloride and EDTA Procedure A. Gel Shift Reaction 1. Prepare a 20 l binding reaction by combining the following ingredients: 4 l of 5X Binding Buffer 0.2 l of 0....