生物信息学技术

Big Dye Protocols and Notes - Cosmid BAC BAC Fosmid TemplatesHi all Over the past two months we have been testing various reaction conditions for the ABI Big-Dye Terminators with double-stranded DNA t ...

BAC End-Sequencing(Diana Bocskai)For every 4 mls of culture dissolve the BAC DNA pellet in 40 µl of water. for example: Usually each BAC is grown in 20 mls LB/CM total then is dispensed into one Autog ...

Introduction The most important aspect of our cloning vectors is that they arebased on the E. coli F-factor replicon. It allows for strict copynumber control of the clones so that they are ...

Protocol for Construction of BAC LibrariesThe bacterial artificial chromosome cloning (BAC) system is emerging as the system of choice for construction of plant genomic libraries with average in ...

YAC TRANSFORMATION OF C. ELEGANS USING TOTAL YEAST GENOMIC DNA 15(1) 1. PREPARING YAC (+ Yeast) DNA BY CsCl PURIFICATION.(Adapted from Stiles (1983) Methods in Enzymology 101: 296-297) Grow 3ml cultu ...

Megabase DNA IsolationMegabase-size DNA isolation from plantsTo construct large insert DNA libraries in BAC and YAC vectors methods must be developed to isolate very high molecular weight DNA - megaba ...

Protocol for hybridization of high density arrayed Bacterial Artificial Chromosome nylon filter blots with 100 PCR isolated Unigene cDNA inserts pooled in a combined probeIsolation and purification of ...

Gel-purified agarased YACs for yeast transformationProtocol from Andreas Gnirke (formerly of the Olson lab) with modifications by Anja van Brabant (Fangman/Brewer lab)Materials:1. Yeast DNA plugs at 4 ...

cDNA Library Screening Protocol - ZAPIIProcedure:1) Streak XLI-Blue MRF' from frozen stock on an LB-tetracycline plate. Incubate plate overnight at 37°C. Pour NZY plates and store at room temp. to dry ...

Construction of a BAC libraryA. Generation of large DNA fragments from megabase-size DNA for BAC cloning Once high molecular weight (HMW) DNA has been prepared it must somehow be fragmented and DNA in ...

SOLUTIONS FOR BAC LIBRARY CONSTRUCTION 10X Homogenization Buffer (HB) stock: (1 liter)IngredientAmountFinal ConcentrationTrisma base12.1 g0.1 MKCl59.7 g 0.8 MEDTA37.2 g0.1MSpermidine-HCL2.55 g10 mMSpe ...

Screening a cDNA Libraryfor use with HybriZAP zebrafish cDNA libraries ObjectivecDNA library screening allows detection of expressed genes for subsequent cloning and sequencing. Selecting a LibraryCur ...

COLONY HYBRIDIZATION 1) CUT 4 PIECES OF 3MM WHATMAN PAPER AND PLACE EACH ONE IN A SEPARATE CONTAINER(A CAFETERIA TRAY WILL PROBABLY WORK PERFECTLY). 2) SATURATE EACH OF THE 4 PIECES WITH ONE OF THE FO ...

Choice of bacterial strain: The bacterial E. coli strains we use when plating out cDNA and genomic libraries are normally LE392 and NM538 but some times we also use XL1-blue cells. Streak out bac ...

Outline:Bacteriophage lambda is a linear double stranded DNA approximately 50 kB in size. The two sticky ends help the phage to recircularize after entering the bacterial host cell. A cDNA library is ...

Experimental Protocol for cDNA Library ConstructionIdentify appropriate celltype over-expressing corresponding gene.Find out if transcription can be stimulated further eg by induction (note: the highe ...

Genomic DNA libraries Size of some genomes and chromosomes:Comparative Sequence Sizes(Bases)(yeast chromosome 3)350 ThousandEscherichia coli (bacterium) genome4.6 MillionLargest yeast chromosome now m ...

Packagene® Lambda DNA Packaging SystemThe Packagene® Lambda DNA Packaging System is derived from the unique one-strain host system (Rosenberg). The System is easy to use because it contains all the co ...

Genomic Cloning Technical ManualAn optimal strategy for genomic cloning should meet three requirements: 1) a maximum number of recombinants should be obtained with a minimal background of nonrecombina ...

DNA Isolation From BAC & PAC Clones This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic ...