DNA测序

作者：佚名    实验频道来源：中华基因网    点击数：    更新时间：2004-6-8

DNA测序（主要内容如下） ·         Sequencing Gel Preparation ·         Preparation of Templates  ·         DNA Sequencing by the Dideoxy Method ·         DNA Sequencing by the Chemical Method ·         Dye Terminator DNA Sequencing ·         Construction of Nested Deletions for Sequencing ·         PCR Sequencing ·         Recipes ·         Q & A Posted in Molecular Biology Method Forum Sequencing Gel Preparation ·         Sequencing Gel Prep (NWFSC) Gel preparation, buffer, electrophoresis... ·         Sequencing Gel Prep (NWFSC) Wedge gel performance without pouring wedge gels ·         Sequencing Gel Prep (Promega) ·         Buffer Gradient Sequencing Gel (Bowtell Lab) ·         Sequencing Gel Prep ·         Buffer Gradient Sequencing Gels (PMCI Research) Preparation of Templates  ·         Preparation of Template DNA for Sequencing (Promega) ·         PCR Product Sequencing (Genetics Resources CORE Facility at Johns Hopkins U) Detailed method for preparing PCR products for direct sequencing ·         Plasmid DNA Preparation for Sequencing (Genetics Resources CORE Facility at Johns Hopkins U) ·         Phage and Cosmid DNA Preparation for Sequencing  (Genetics Resources CORE Facility at Johns Hopkins U) DNA Sequencing by the Dideoxy Method ·         Double Stranded DNA Sequencing (Dideoxy) Double Stranded DNA Sequencing (Dideoxy) ( (Bowtell Lab) Sequencing of Double Stranded DNA Using Dideoxy Chain Termination (Donis Keller Lab) This method is used to sequence double stranded DNA, such as a plasmid insert or purified PCR product. The dideoxy chain termination (or enzymatic) method of DNA sequencing involves the in vitro synthesis of a DNA strand by a modified bacteriophage T7 DNA polymerase (SequenaseR/USB) using a single stranded DNA template. ·         DNA Sequencing Reaction (Lazo Lab) ·         Old Fashioned Sequencing(Brent Graveley) ·         DNA Sequencing Using an End-Labeled Primer (Promega) ·         DNA Sequencing  Using Direct Incorporation (Promega) ·         Double Stranded DNA Sequencing (Dideoxy) (PMCI Research) DNA sequencing (Roe Lab)                             i.            Bst-catalyzed radiolabeled DNA sequencing                         ii.            Radiolabeled sequencing gel preparation, loading, and electrophoresis                     iii.            Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers                         iv.            Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions                             v.            Sequenase[TM] catalyzed sequencing with dye-labeled terminators                         vi.            Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer                     vii.            Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers                 viii.            cDNA sequencing based on PCR and random shotgun cloning DNA Sequencing by the Chemical Method ·         Preparation of G+A Marker Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing reaction and works fine. Dye Terminator DNA Sequencing Dye Termination Sequencing of dsDNA. (Gimila Lab)     ·         ABI Dye Primer Reactions using FS (Whitehead Institute/MIT) ·         ABI Dye Primer Reactions using ThermoSequenase(Whitehead Institute/MIT) ·         ABI Dye Primer Reactions Home Brew(Whitehead Institute/MIT) ·         ABI T7 Terminator Reactions(Whitehead Institute/MIT) ·         ABI AmpliTaq Dye Terminator Reactions(Whitehead Institute/MIT) ·         Dye Terminator Cycle Sequencing (PMCI Research) ·         ALF Sequencing with Fluorescein Labelled Nucleotides (PMCI Research) Construction of Nested Deletions for Sequencing ·         Nested Deletions (Bowtell Lab) Nested Deletions using exonuclease-III and mung bean nuclease ·         Site-directed Mutagenesis (Promega) For generation and selection of oligonucleotide-directed mutants ·         Deletion Mutagenesis (Promega) For construction of plasmid or M13 subclones containing progressive unidirectional deletions of  inserted DNA, Including methods for DNA preparation, vector digestion, exonuclease deletion, ligation,  transformation... ·         Unidirectional deletions (erase-a-base) (Crawford Lab) PCR Sequencing ·         PEG Purification of PCR Products (GSCWU) This protocol can be used instead of EXO/SAP for removing excess primers and nucleotides from PCR products before cycle-sequencing ·         Purification and Sequencing of PCR Product (Donis-Keller lab) This method is used to sequence directly a specific PCR product after large scale amplification. The reaction should be carried out in the usual fashion, except that after optimization of annealing temperature and other conditions in the 5 µl volume, a large scale 50 µl (10X) total reaction volume should be used to generate sufficient PCR product for sequencing. ·         PCR Product Sequencing (Genetics Resources CORE Facility at Johns Hopkins U) Detailed method for preparing PCR products for direct sequencing 96 Well Plate Purification of PCR products Purification with Sepahcryl S-300 or S-500 - in filter bottom plates  96-well silent screen plate, Purification with Guanidine-HCl in MES buffer - in glass filter plates Purification with Guanidine-HCl in KAc buffer    Preparation of PCR product for direct sequencing (Robert H. Cruickshank) Chloroform Extraction Modified Microcon-100 protocol for purification and concentration of DNA from PCR product Measurement of DNA concentration  Recipes Recipes for Sequencing (Sequencing Group, UW) Recipes for gel solutions, loading buffer...  处理 SSI 文件时出错