DNA测序
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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)
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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
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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)
・ 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
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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
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Recipes for Sequencing (Sequencing Group, UW)
Recipes for gel solutions, loading buffer...