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47 Cards in this Set
- Front
- Back
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What are most methods for bacterial transformation based on?
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The observation that bacteria treated with ice-cold solutions of CaCl2 and then briefly heated, could be transformed by bacteriophage lambda DNA as well as plasmid DNA.
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What does the CaCl2 do?
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Induces a transient state of "competence" in the recipient bacteria
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What does the CaCl2 induced transient state allow?
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They are able to take up DNA from a variety of sources.
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How many transformed colonies do bacteria treated with Cacl2 typically yield?
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10^5-10^6 transformed colonies/ug of supercoiled plasmid DNA
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By how much can one increase the efficiency of transformation?
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100-1000 fold.
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How can one achieve this efficiency?
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By exposing "improved" strains of E. coli to combinations of divalent cations for longer periods of time
-treating with DMSO -treating with reducing agents (DTT or Beta-mercaptoethanol) or hexamine cobalt chloride |
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What is a divalent cation?
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An ion with a net charge of positive 2
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What was the efficiency of transformation with 50 ng of plasmid DNA when cells were suspended only in LB broth?
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0
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What was the efficeincy of transformation when the cells were treated with MgCl2? With beta-mercaptoethanol?
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Around 5 000 colonies for cells that werent treated with 2ME and around 10 000 for cells that were treated with 2ME
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What was teh efficiency of transformation for cells treated with both MgCl2 and CaCl2?
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Around 400 000 for cells that were not treated with 2ME and around 500 000 for cells that were treated with 2ME
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How can one be sure that their cells are competent?
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It is possible to purchase frozen competent bacteria from commercial sources
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What are some characteristics of the commercial competent cells?
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Reliable, and have a transformation frequency of 10^8 colonies/ug of supercoiled plasmid DNA
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When are these commercial products used?
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When constructing plasmid libraries of cDNAs synthesized from extremely small amounts of mRNA or mRNA isolated from relatively inaccessible sources
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When are high transformation efficiencies NOT required?
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When cloning PCR fragments into a plasmid or subcloning a cDNA from one plasmid to another
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How are "home-brewed" cells generated?
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by treating bacterial cells with CaCl2
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In addition to chemical methods, what is another way to transform bacteria?
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High voltage electroporation
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How was the high voltage electroporation technique initially utilized?
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Introducing DNA into eukaryotic cells
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What are the average transformation efficiencies when using high voltage electroporation techniques?
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10^9-10_10 transformants per ug of DNA
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What are some parameters that can be adjusted to optimize the technique of electroporation?
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The stength of the electric field, the length of the electric pulse, and the concentration of DNA.
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For what parameter definitions were the highest efficiencies obtained?
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At higher voltages or longer pulses
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What wre these high efficiences offset by?
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A decrease in cell viability
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What is the most important factor in overall transformaiton efficiency in most chemical methods?
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The size of the plasmid DNA
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How does size affect efficiency?
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Smaller plasmids are picked up more efficiently than large plasmids
Supercoiledplasmids enter the cells more readily than relaxed circular DNA or linear DNA |
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When using electroporation, what size of supercoiled plasmids can be transformed?
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25-130 kb can transform with similar molar efficiency
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How are cells prepared for electroporation?
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Cells are grown to mid log phase, chilled on ice, washed with low salt buffers.
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What does the washing with low salt buffers do?
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Reduces the ionic strength of the cell suspension
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At what temperature is electroporation carried out?
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At low temperatures (0-4 degrees celcius)
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Why is electroporation carried out at a low temperature?
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The efficiency drops as much as 100 fold when the experiment is carried out at room temperature
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Typically how are competent cells prepared?
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From exponentially growing cells from a single colony
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What type of dilution of an overnight culture of E coli will reach its mid exponential growth after 2.5-3 hours?
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A 1:100 dilution
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What are the parameters for this exponential growth?
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37 degrees temperature in NON-selective LB broth
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What is used to monitor the cell density of the solution?
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The OD 600
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What is the OD 600 at mid exponential growth?
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0.45 to 0.55
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What happens after the bacteria are grown to mid exponential phase?
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They are chilled and treated with 0.1 M CaCl2 for up to 24-48 hours on ice
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What is added in our experiment to increase transformation efficiency?
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A reducing agent
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At what concentration and volume can plasmid DNA be added to the cells?
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At a concentration of 1-50ng in a volume of 10 ul or less
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Why are the bacteria warmed to 42 degrees after being incubated on ice for 30 min?
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To facilitate DNA entry into the cells
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Why are the cells incubated at 37 degrees for 60 min?
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This is a recovery period that will allow the transfored cells to express beta-lactamase that will confer antibiotic resistance to the transformed cells, allowing the bacteria to grow on selective plates or medium
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What are the three distinct phases of E coli growth?
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The lag phase (or pre-exponential phase)
THe exponential phase The plateau phase |
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What is the lag phase characterized by?
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A slow rate of cell division
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What does the exponential phase represent?
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The period at which cell division is maximal
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At what rate do cells growing in culture typically double?
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40-60 minutes
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What happens in the plateau phase?
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The growth rate is reduced as a result of nutrient depletion in the medium and high cell density
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What does incubation of the cells in a sucrose rich medium do?
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This will allow them to recover from the previous heat shock and to synthesize enough beta-lactamase to resist ampicillin selection
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What efficiency of transformation defined by?
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The number of colonies per ug of plasmid DNA
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At what voltage is electroporation performed?
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2.5kV
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What is the name of the E coli strain used in this exercise?
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DH5 alpha
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