Storage of gene libraries as glycerol stocks in 1536-well plates

Abstract

Individual clones from genomic and cDNA libraries are now commonly stored as glycerol stocks in the wells of microtitre plates. The most common format of microtitre plate used for this purpose is the 384-well plate. As a 100,000 clone library requires 260 384-well plates and, normally, several copies of the library are stored, a considerable amount of -80 "C freezer space is required for the storage of significant numbers of libraries.One way of reducing the storage space required is to increase the number of wells in each microtitre plate, and this has lead to the development of the 1536-well plate. However, the volume of medium contained in each well of a 1536-well plate (8 1. 1) is considerably less than in a 384-well plate (50 1. 1) . This raises concerns about the viability, following storage, of clones in 1536-well plates.We have demonstrated that the viability of clones stored in 1536well plates, following storage at -80 "C, is equivalent to that of clones stored in 384-well plates.The T341C mutation in human NAT2 gene is present in many populations, and is associated with the slow acetylator phenotype. A new PCR-RFLP method is described for the detection of this functionally important mutation. The method is based on mutagenesis-directed PCR to create an Nco I site for the wild-type allele. Using this method, w e have genotyped 110 unrelated Omani subjects, and assessed the accuracy of results by repeating the analysis with a complementary DdeIbased PCR-RFLP method. There was 100 % concordance between the two methods. The allele frequency of the T341C mutation for Omani population was determined as 0.44, and compared with the reported values for other populations. This is the first report describing the allele frequency of the T341C mutation in an Arab population. DenmarkRecent studies of the combination of Biacore and mass spectrometry (BIA/MS) have used antibodies (1-3), DNA (2) or high affinity tagbinding molecules like streptavidin (3) as the immobilized receptor and peptides or proteins as ligands. The two described strategies for the combination of BIA/MS are, (i.) define MALDI-TOF directly on the sensor chip after binding of the ligand, and (ii.) elution of the ligands from the Biacore instrument followed by mass spectrometric analysis. Since Biacore has become an important tool in low molecular weight compound analysis, we have now investigated the application of the BIA/MS combination to studies of low molecular weight compounds, e.g. drugs. HIV-protease 1 (22kDA) was immobilized to the sensor chip and the inhibitors Saquinavir (670 Da) and Indinavir (613 Da) were captured. We demonstrate that the inhibitors can be selectively captured from mixtures of low molecular weight compounds and mass analyzed after elution from the Biacore instrument. To confirm the identity of the eluted compounds, structural information was obtained by MALDI-TOF PSD as well as electrospray MS/MS in an iontrap mass spectrometer. Furthermore, a competition test with Saquinavir and Indinavir resulted in detection of both d...