AgarPlatesforSelectionofClonesinBacteria
Cloning of PCR productsStocks:LB Agar: Luria Broth after Lennox:per LiterTryptone10 gYeast Extract5 gSodium Chloride5 gBact. Agar15 g pH 7.0Autoclave and store sterile at room temperature Stock solutions: Store all frozen in aliquots at -20C (can be stored in dark at 4C but -20 is usually as simple!)X-Gal: 20 mg/ml in Dimethylformamide (DMF) IPTG: 100 mM Ampicillin: 100 ug/ml (microgram......閱讀全文
Agar－Plates－for－Selection－of－Clones－in－Bacteria
Cloning of PCR productsStocks:LB Agar: Luria Broth after Lennox:per LiterTryptone10 gYeast Extract5 gSodium Chloride5 gBact.? Agar15 g?pH 7.0Autoclave
Preparation－of－Agar－plates
Prepare media and add 1.5 agar before autoclaving it (15g per liter).?After autoclavation, cool the media in a 55 degree waterbath. Do not allow?the s
Transformation－of－Electrocompetent－E.－coli－with－Blue/White－selection
Desalt DNA template by EtOH precipitation in NaOAc followed by at least 2x washes with 70% EtOH.? Resuspend in 5 - 15 μL of sterile H2O.Rinse cuvettes
Preparing－Antibiotics－Stock－Solution－and－Ampicillin－Agar－Plates
AMPICILLIN?Beta-lactam-antibiotics are not very stable when dissolved. Slow but steady degradation happens even when frozen to -20?°C. Therefore comme
Expression－Library－Screening－(Procaryotic)－Using－APFusion－Proteins
Outline:Bacteriophage lambda is a linear double stranded DNA, approximately 50 kB in size. The two sticky ends help the phage to recircularize after e
Noble－Agar－Assay
DescriptionCancer cells do not show anchorage and contact inhibition of growth. To assess the anchorage and contact independent growth of cells, noble
Phage－Titer
IntroductionLambda phage is a commonly used vector for transgenes. Its very high rate of infectivity makes it ideal for creating large numbers of clon
Colony－Hybridization
ProcedurePrepare serial 10-fold dilutions of transformed bacteria in LB and spread 100 μL onto LB/Amp plates, as described in the Electroporation prot
土壤細菌的分離與純化
一 教學要求 通過從土壤中分離純化細菌 ?，初步掌握微生物的分離純化方法和無菌操作技術。 二 實驗原理 - 常用的分離純化方法 microorganisms exist in Nature ?as mixed populations. However, to study microo
Streptomyces:Protocols/Spore－Prep
Spore Prep - Inoculating & HarvestingDescription?A spore prep is a method of preserving a sporulating strain of Streptomyces. The stock is stored in 2
細菌培養
Preparing Overnight Bacteria Culture?(LaboratoryExperiments.com)This is a basic procedure for high school students and useful for those who are new to
Transformation－Protocol－for－Arabidopsis
Transformation Protocol for Arabidopsis – AbbreviatedGerminate seed in pots↓ 4 weeksStreak bacteria onto YM/MinA↓ 2-3 days 28°CSpray/dip bacterial sus
ELECTROPORATION－OF－ES－CELLS－AND－ISOLATION－OF－H/R－CLONES
Need 1.5-2 x 107 cells from a 2 day culture.1. Cells are harvested as normal, washed x 1 in PBS then taken up at conc. of 1.2 x 10 7 cells/ml in cold
Standard－Operating－Procedures－for－T1Phage－Testing－Assay
I. Introduction:This assay uses a lawn of phage-susceptible?E. coli?(DH10B) embedded in a layer of agarose. This top agarose lays on a bed of standard
Expression－L19－using－Pichia－pastoris
Pichia pastoris is a methylotropic yeast used to express high amounts of protein. Secreted expression is achieved with a cleavalable faktor. To yield?
Yeast－Media
YEPD (non-selection)-1% yeast extract-2% peptone-1.5% agar (if needed for plates)After autoclaving add glucose to 2% by adding 100 ml of 20% solution
Preparation－of－Bacterial－Proteins－for－Analysis－by－2DPAGE
The following protocol has been developed for preparing soluble bacterial proteins in a form suitable for analysis by 2D-PAGE. The procedure was princ
Preparing－Lambda－DNA
Preparing Lambda DNA1- Coliphage lambda DNA is a widely used vector for recombinant DNA. The middle third of its 48,000 bp contains no genes required
Sterile－Technique
Good sterile technique is the first and most important step in insuring consistent results when employing recombinant DNA and protein expression techn
Cosmid－Cloning:－Cell－preparation,－DNA－packaging,－and－Cell－Transfection
Cosmid Cloning: Cell preparation, DNA packaging, and Cell TransfectionProtocol taken from Stratagene's Gigapack packaging extracts instruction man
Easy－Way－to－Clone－Genes－From－a－Phage－Library
Easy Way to Clone?The protocol is oriented towards a C. albicans genomic library I made in Lambda Zap II on 7/97.The overall sequence of events is:??
無菌化技術
Sterile TechniqueGood sterile technique is the first and most important step in insuring consistent results when employing recombinant DNA and protein
Lactobacillus－culture
OverviewGeneral overview and guidelines on how to grow up a culture of LactobacillusMaterialsMRS broth (difco)MRS agar (difco)anaerobic conditionsProc
Screening－Bacterial－Colonies－Using－Xgal－and－IPTG:－αComplementation
實驗概要? ? ? ? α-complementation occurs when two inactive fragments of E. coli β-galactosidase associate to form a functional?enzyme. Many plasmid
Layered－plates
General InformationThe cells being plated are sandwiched between layers of cell-free agar. The submerged cells grow into smaller colonies and many mor
Pouring－Plates
1. For rich media, weigh out appropriate ingredients and place into a flask. Add water until appropriate volume. Use a flask at least 2 times larger t
Bacterial－cell－culture
MaterialsGlass culture tubes with metal caps and labelsGrowth medium, from media room or customizedGlass pipette tubesParafilmEquipmentVortexerFireboy
DNA轉化實驗指導3
6.?????Simultaneous digestion of the pUC vector with both enzymes in the presence of 3 units of Shrimp Alkaline Phosphatase (Amersham BioSciences)?in
Generating－stable－cell－lines－in－HEK293
Generating stable cell lines in HEK293Prior to transfection, it is recommended that you linearize your pcDNA gene construct. Linearizing will decrease
Generating－stable－cell－lines－in－HEK293
Generating stable cell lines in HEK293Prior to transfection, it is recommended that you linearize your pcDNA gene construct. Linearizing will decrease