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Bacterial TransformationBacterial Transformation
Broad and Long Term ObjectiveBroad and Long Term Objective
To characterize a single clone from an To characterize a single clone from an Emiliania huxleyiEmiliania huxleyi cDNA library using cDNA library using sequence analysissequence analysis
Research PlanResearch Plan
Preparation of Competent Cells and Bacterial Transformation
Growth of Transformant and Plasmid MiniPrep
Cycle Sequencing
Sequence analysis
Today’s Laboratory ObjectivesToday’s Laboratory Objectives
1.1. To prepare competent cellsTo prepare competent cells
2.2. To perform a plasmid transformationTo perform a plasmid transformation
3.3. To quantify transformation efficiencyTo quantify transformation efficiency
DefinitionsDefinitions
““Competency” refers to the Competency” refers to the abilityability to take up foreign DNA to take up foreign DNA
some bacterial cells are naturally competent and have special some bacterial cells are naturally competent and have special proteins that are involved in DNA uptake and integration into the proteins that are involved in DNA uptake and integration into the chromosomechromosome
artificially induced competent is where DNA uptake is induced by artificially induced competent is where DNA uptake is induced by chemical or physical meanschemical or physical means
Definitions Con’tDefinitions Con’t
““Transformation” is the Transformation” is the uptakeuptake of free foreign of free foreign DNA into the cellDNA into the cell
Components of a Components of a Transformation SystemTransformation System
1.1. Mode of DeliveryMode of Delivery
2.2. Selectable MarkerSelectable Marker
3.3. Propagation of Clonal IsolatesPropagation of Clonal Isolates
1.1. Chemical treatmentChemical treatment
2.2. ElectroporationElectroporation
3.3. Microparticle GunMicroparticle Gun
Nucleic Acid Delivery Methods
Chemical TreatmentChemical Treatment
Cold Shock with high concentrations of calcium chloride Cold Shock with high concentrations of calcium chloride and magnesium chlorideand magnesium chloride
ElectroporationElectroporation
Electric Shock Opens Pores in Cell WallElectric Shock Opens Pores in Cell Wall
Microparticle BombardmentMicroparticle Bombardment
Shoots projectiles of gold or tungsten coated with DNA or RNA into Shoots projectiles of gold or tungsten coated with DNA or RNA into cells using an inert gascells using an inert gas
Generally used with EucaryotesGenerally used with Eucaryotes
Selectable MarkerSelectable Marker
1.1. Antibiotic Resistance MarkersAntibiotic Resistance Markers
2.2. Complementation using Essential Metabolic Enzymes Complementation using Essential Metabolic Enzymes
Map of Positive Control VectorMap of Positive Control Vector
Map of Parent Vector pMAB58 of Map of Parent Vector pMAB58 of Experimental cDNA CloneExperimental cDNA Clone
pMAB58
7577 bps
1000
2000
3000
4000
5000
6000
7000
SwaIPpuMI
AhdI
AlwNI
Asp718IKpnIApaIBsp120I
StyIBsmI
PacIBsrGI
BsaBINspV
RsrIIEco47IIIBseRI
SexAIMluI
EcoRIXbaI
BsmIBsrGISmaI
BsaBIEcoRI
NotIMluI
BsrGIAatII++
SacII
BamHI
DraIII
Bsu36IXbaI
BspEISnaBI
ARS4/Cen6
Amp
ori
pADHNLS
ADattB1
ccdB
attB2
Ter ADH
pT7
F1
TRP1
Ampicillin as a Seletable Marker
When plated on media containing ampicillin, transformants harboring plasmids containing an ampicillin resistance gene will survive. Untransformed cells lyse in the presence of ampicillin.
Propagation of Clonal Isolates
Plasmid TransformationPlasmid Transformation
ControlsControls
+ Control= tests the competency of cells+ Control= tests the competency of cells
- Control= tests the efficacy of selectable marker- Control= tests the efficacy of selectable marker
Transformation Efficiency:Transformation Efficiency:
Number of transformants/ng of DNANumber of transformants/ng of DNA Count number of colonies on plate after overnight growthCount number of colonies on plate after overnight growth Determine the proportion of the total transformation that was Determine the proportion of the total transformation that was
plated (ie, volume plated/total transformation reaction volume)plated (ie, volume plated/total transformation reaction volume) Express in terms of the amount of DNA used in the reactionExpress in terms of the amount of DNA used in the reaction
Number of transformants x (total transformation volume/volume plated)Number of transformants x (total transformation volume/volume plated)
ng of DNA used in transformationng of DNA used in transformation
Next WeekNext Week
1.1. Perform a Plasmid Miniprep Perform a Plasmid Miniprep
2.2. Quantitate Plastmid IsolatedQuantitate Plastmid Isolated
3.3. Determine the Size of the PlasmidDetermine the Size of the Plasmid