Customer Support - Application Notes

Introduction:

Users of Big Dye version 3 chemistries have reported occasional dye blobs on sequence images even after cleanup of reactions using spin columns or plates. The blobs are apparently due to aggregation of the dyes. ABI has released a modification of the cleanup protocol supplied with the version 3 chemistry. The recommendation is to add 1/10th volume of 2.2% SDS to the sample, mix, heat to 95 degrees C for 5 minutes, cool to ambient temperature and spin briefly to collect sample in bottom of tube.. The sample is then added to the spin column as previously described. This additional step is also recommended for samples processed using Centri·Sep 96 plates.

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Nucleic acid hybridization with labeled probe sequences is a well known analytical tool used in molecular and cell biology. The utility of hybridization has been greatly increased by the development of in vitro methods for incorporation of label (either isotopic or nonisotopic) into the nucleic acid probe molecules. One of these methods is nick translation, where nucleotides are incorporated into double stranded DNA by DNA Polymerase 1 at sites where the molecule has been nicked by DNAse 1. Nucleotide mixes containing a label 32P-dATP or Fluorescein-dUTP) provide the pool of nucleotides which DNA Polymerase 1 will incorporate into the double stranded DNA molecule. The excess labeled nucleotides are then separated from the finished, "labeled" DNA.

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Introduction:

The high salt concentrations present in ligation mixes are known to reduce electroporation transformation efficiency. Recommendations to reduce salt concentration prior to electroporation include float dialysis, centrifugal ultrafiltration, and dilution. Each of these are either time consuming, not completely effective (reducing the transformation efficiency), or worse, result in significant DNA loss.

A Centri-Spin 20 protocol for desalting ligation reactions eliminates salts and co-factors while recovering >85% of the DNA. Electroporation transformation efficiencies are increased 2-fold.

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Introduction:

We have developed a spin column miniprep protocol for the purification of BAC DNA. No organic extractions are required. Typical yields between 0.6 and 1.0 µg are observed from an overnight 3-5 mL culture. A discussion of the data and results depicting the quality of BAC DNA purified is presented. Alternative protocols for preparation of BAC DNA from 25-250mL cultures are also presented. Observations of some factors that affect BAC yield will be discussed.

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