Sunday, February 01, 2004 Description High GC content can prohibit the PCR reaction due to improper denaturing of the template or annealing of the primers. This minor spin on a common protocol is simple but works incredibly well. Procedure...
Thursday, December 04, 2003 Description Tail PCR Procedure Amplification of flanking sequences To determine flanking sequences of RDA subtraction products in the genome, a two-step PCR reaction technique can be applied using a biotinylated...
Friday, November 21, 2003 Description OLIGO PURIFICATION on acrylamide gels Procedure GEL PURIFICATION OF OLIGONUCLEOTIDES 1. Quantitate crude oligo solution via UV spectrophotometry. Assume 1.0 A260 unit is equal to 33 ug/ml. Dry down 100...
Friday, November 21, 2003 Description PRIMER LABELLING FOR PRIMER EXTENSION ASSAY and PRIMER EXTENSION Procedure Mix in a 20ul final volume: 2ul 10x kinase buffer 1ul oligonucleotide primer (5pmoles/ul) 200uCi 32P-gATP (crude, ICN, 7000Ci/...
Friday, November 21, 2003 Description This technique exploits the fact that a protein bound to a specific DNA sequence will interfere with the digestion of that region by the endonuclease DNAaseI. In summary, an end-labelled DNA probe is i...
Friday, November 21, 2003 Description Nested Deletions using exonuclease-III and mung bean nuclease Procedure ExoIII has a 3' to 5' exonuclease activity which must initiate on blunt ended DNA or DNA with a 5' overhang. 1. A nested series o...
Friday, November 21, 2003 Description Nested Deletions using exonuclease-III and mung bean nuclease Procedure ExoIII has a 3' to 5' exonuclease activity which must initiate on blunt ended DNA or DNA with a 5' overhang. 1. A nested series o...
Friday, November 21, 2003 Description In vitro mutagenesis using Altered Sites Procedure A. Isolation of Single Stranded DNA 1. Transform plasmid to be mutagenised into JM109. JM109 is endA1, recA1, gyrA96, thi, hsdR17(rk-, mk+), relA1, su...
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...