PurifyingLargeE.coliRestrictionFragmentsfromPulsedFieldGels
DNA PreparationE. coli chromosomal DNA is prepared following the method of Heath et al. ( J. Bacteriol., 174, 1992). Cells are embedded in agarose, then treated with deter gents and enzymes which remove the cell wall, proteins and other cellular material. The purified chromosomal DNA remains embedded in the agarose plug.Preparation of cell plugs1-Grow an overnight culture of E. coli MG1655 in L broth. The OD600 of an......閱讀全文
Purifying－Large－E.－coli－Restriction－Fragments－from－PulsedField－Gels
DNA PreparationE. coli chromosomal DNA is prepared following the method of Heath et al. ( J. Bacteriol., 174, 1992). Cells are embedded in agarose, th
DNA的酶學操作
DNA的酶學操作DNA Modifying Enzymes?(Michael Blaber)Introduction to bacterial restriction/modification system. It provides very useful background knowledge
Southen雜交
Southern雜交One important thing for transfer:the weight of the object resting on top of the blotting apparatus should not exceed the weight equal to a 5
Construction－of－BAC－Libraries：Construction－of－a－BAC－library
Once high molecular weight (HMW) DNA has been prepared it must somehow be fragmented and DNA in the desired size range isolated. In general, as the de
DNA抽提
DNA抽提（主要內容如下）·???Working with DNA·???DNA Extraction from Bacteria and Other Organisms·???DNA Extraction from Cell and Tissue·???Mitochondria DNA Isola
DNA電泳
DNA電泳（主要內容如下）??Preparation of Agarose Gel and Electrophoresis??Extraction of DNA From Agarose Gel??Extraction of DNA from Acrylamide Gels??DNA Marker?
Pulse－Field－Electrophoresis
Manipulating and analyzing DNA are fundamentals in the field of molecular biology. Indeed, separating complex mixtures of DNA into different sized fra
Construction－and－Manipulation－of－LargeInsert－Bacterial－Clone－Libraries
Acknowledgements?The organizer of the workshop acknowledges Dr. Murray Milford, Professor and Interim Head, and Dr. Mark Hussey, Professor and Interim
細菌的核酸抽提
DNA Extraction·?????????DNA Extraction from Bacteria?(Julie B. Wolf,UMBC)Phenol/chloroform method·?????????DNA Extraction From Bacteria (Triton Method
Denaturing－Gradient－Gel－Electrophoresis－(DGGE)
Purpose:Denaturing gradient gels are used to detect non-RFLP polymorphisms. The small (200-700 bp) genomic restriction fragments are run on a low to h
DNA克隆
DNA克隆（主要內容如下）·?????????General Procedure·?????????PCR Cloning·?????????Subcloning·?????????ET Cloning·?????????Vector Preparation·?????????Ligation Re
DNA－Purification－from－Agarose－Gels
1. Separate DNA fragments in an agarose gel cast with 0.5 mg/mL Ethidium bromide. Locate bands with a hand-held long-wave UV lamp.2. Slice the gel wit
酵母人工染色體
·?????????Easy YAC Preparation Method?(Andrew Davies,Shaw lab)·?????????Screening YAC libraries?(Donis Keller Lab)This is a method for screening YAC l
BAC－DNA分離方法－Isolation－of－BAC－DNA－from－Largescale－Cultures
Isolation of BAC DNA from Large-scale CulturesJoseph SambrookPeter Maccallum Cancer Institute and The University of Melbourne, AustraliaDavid W. Russe
Basic－procedures－for－bacteria－culture2
E. Elution of DNA fragments from agaroseDNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first develo
重組DNA的分離、克隆與測序實驗手冊2
C. Restriction digestionRestriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restrict
Determining－the－Direction－of－Replication－Fork－Movement
For an in-depth review of the method, see Friedman, K. and B. Brewer (1995) Analysis of replication intermediates by two-dimensional agarose gel elect
2d2D電泳
For an in-depth review of the method, see Friedman, K. and B. Brewer (1995) Analysis of replication intermediates by two-dimensional agarose g
PCR的下游應用
·?????????Agarose Gel Electrophoresis of PCR Products?(Robert H. Cruickshank)·?????????Agarose Gel Electrophoresis of PCR Products?(Immunology Resourc
Maxiprep－of－plasmid－DNA－from－E.－coli
IngredientsIngredients are per culture; make enough for one extra culture to allow for pipetting error).150μL sterile 50% glycerol1mL TEG (25mM Tris-C
Sauer:RNA－Purification－from－E.－coli
My Experience Purifying RNA from E. coliRegarding RNA extraction, there is a horrible tendency of people to use kits for RNA extraction with bacteria.
Ligation－Optimization
The following protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independent
Basic－procedures－for－bacteria－culture1
A. Phenol extraction of DNA samplesPhenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated
Cloning－PCR－products－using－TA－vectors
Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D.?*Methods and reagents is a unique monthly column that highlights current discussions in
Edman－Sequencing－of－Proteins－from－2D－Gels
The Western blotting/sequencing technique using polyvinylidene difluoride (PVDF) membrane is one of the most popular technique for Edman sequencin
PCR的下游應用
??????????Agarose Gel Electrophoresis of?PCR?Products(Robert H. Cruickshank)??????????Agarose Gel Electrophoresis of PCR Products(Immunology Resource)
DNA轉化實驗指導2
1B.??Cloning?1.?????A caveat on dephosphorylation: the most common reason for failure to obtain colonies is a result of adding too much BAP or CIP to
Maxiprep－of－plasmid－DNA－from－E.coli－protocol
Solutions/reagents:LB broth + selective marker50% sterile glycerolTEG(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)20 mg/ml lysozyme10% SDS4M NaOHautoclaved
General－Cloning－Protocols
Large Scale Preps:?(See Large scale plsasmid prep protocol for more details)Cultures: Inoculate a 5 mL LB/Amp (50 - 100 μg/mL) culture in early a.m. w
ELECTROPHORESIS－OF－DNA－IN－AGAROSE－GELS
ELECTROPHORESIS OF DNA IN AGAROSE GELSA). AGAROSE CONCENTRATIONS:???????Use 0.8% agarose (w/v) for high molecular weight DNA fragments, and 1 - 1.2% f