Analyzing Data

Transformation The DNA molecule is hydrophilic (water-soluble) but cell membranes are made of a very hydrophobic lipid bilayer. Two means of artificial transformation commonly used in labs: electroporation and chemical transformation. During electroporation, short bursts of current are passed through a solution containing bacteria at high voltage. The current makes the cell membrane leaky (porous) for a short time, allowing the cells to take up DNA molecules from the solution. In chemical transformation, bacteria are exposed to solutions which alter their cell membranes enough to make the DNA molecules pass through and into the cell. Chemical transformation procedures sometimes also use a heat shock treatment.

Transformation is Inefficient! Most bacteria in the solution WILL NOT become transformed. Identify the needles in the haystack of bacteria. –Antibiotic resistance genes on antibiotic plates –Other forms of selection in yeast like genes for amino acid synthesis.

Chemical Transformation Escherichia coli cells are grown to log phase. Cells are concentrated by centrifugation and resuspended in a solution containing CaCl 2 Exposure to calcium ions renders the cells able to take up DNA, or competent. Plasmid DNA is mixed with the cells and presumably adheres to them. The mixture of DNA and cells is then heat shocked, which allows the DNA to efficiently enter the cells. The cells are grown in nonselective medium to allow synthesis of plasmid-encoded antibiotic resistance proteins, then plated on antibiotic-containing medium to allow identification of plasmid-containing colonies.

Important Considerations Competent are relatively easy to make fresh and harder to freeze- need to be quick frozen to preserve viability. Competent cells need to be kept in -80° C freezer and kept on ice. They are good for approximately 6 months at the highest efficiency and then gradually lose viability. Competent cells should be used immediately after thawing. Remaining cells should be discarded rather than refrozen.

Factors That Affect Transformation Efficiency DNA Concentration –More DNA is not better- Transformation increases linearly for a while and then plateaus at about 10 ng. We used 0.1 ng DNA in 1 ul. –When comparing competent cells, use the same DNA for all transformations Forms of DNA Linear and single-stranded DNA transforms <1% as efficiently as supercoiled DNA. –Plasmid size- bigger isn’t better. –We used supercoiled DNA and most companies do too to calculate transformation efficiency.

Factors That Affect Transformation Efficiency Purity of DNA –For electroporation, never use more than 1 µl plasmid DNA per transformation. The salts contributed by the preparation can cause low transformation efficiencies. The sample DNA to be transformed by electroporation must be in a low-ionic-strength buffer, such as TE buffer or water. DNA samples containing too much salt will cause arcing at high voltage, possibly damaging both the sample and the machine. –Column-purified DNA is generally free of contaminants that would interfere with chemical transformation. –Contaminants in miniprep DNA can interfere with transformation. Limit the amount of miniprep DNA in a transformation as much as possible and never use more than 5 µl per 50-µl reaction.

Factors That Affect Transformation Efficiency Ligation mixtures inhibit transformation. –Ironic! That is why people start with very competent cells. –Ligase strongly inhibits electroporation, but this effect can be limited by heat inactivating the ligase in the ligation mixture (65°C for 5 min) prior to adding DNA to the cells. Dilute the ligation mixture to dilute the salts.

Factors That Affect Transformation Efficiency Heat Shock –The heat shock step can affect transformation efficiency. Optimal efficiencies are obtained with a thin-wall tube such as a PCR tube or another thin-walled tube (42°C for 45 sec). With thick-walled tubes, such as a microcentrifuge tube, the optimal parameters are slightly different (37°C for 60 sec). When in doubt, it is better to use the 60 second shock at 37°C.

Factors That Affect Transformation Efficiency Length of Time After Transformation –The effect of the expression time depends on the plasmid and the strain. –Longer expression times give higher numbers of colonies, but since they are siblings, the number of transformants is not higher.

Factors That Affect Transformation Efficiency SOC Medium –SOC Medium is highly recommended as the expression medium. SOC Medium gives two-fold better results than LB Medium for chemically competent cells. Selective Plates –Some batches of agar plates are better than others. –Fresh plates are best! –Plating large numbers of cells on selective plates or for extended periods of incubation may allow growth of satellite colonies which are breakthrough colonies because of poor antibiotic stability. –The colonies can be identified by streaking them on the selective agar plates. –If they are true transformants, they will grow. –If they are satellites, they will not grow.

Why this Matters Transformation efficiency is a measure of the amount of cells within the bacterial culture that are able to take up DNA molecules. For some molecular biology projects, such as cloning and subcloning, high transformation efficiency is not critical. However applications such as construction of genomic libraries require that the bacteria have very high transformation efficiency.

Controls We are going to calculate transformation efficiency by counting bacteria on our plates Count the CPlasmid plates # of colonies. Count blue and white separately Average the 1/10 dilutions colony numbers.

Blue-White Cloning The is a molecular technique that allows for the detection of successful ligations in vector-based gene cloning. Works in bacteria plasmids, phage cloning and yeast. DNA is ligated into a vector. The vector is then transformed into competent cells. The competent cells are grown in the presence of X- gal. If the ligation was successful, the bacterial colony will be white; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation, without the need to individually test each colony.

Blue-White Cloning X-gal is a colorless modified galactose sugar that is metabolized by β-galactosidase to form an insoluble product (5-bromo-4 chloroindole) which is bright blue. Isopropyl β-D-1-thiogalactopyranoside (IPTG), functions as inducer of the Lac operon, and is used to enhance the blue color, although it isno always needed. The hydrolysis of colorless X-gal by the β-galactosidase causes the characteristic blue color in the colonies; it shows that the colonies contain vector without insert. White colonies indicate insertion of foreign DNA and loss of the cells' ability to hydrolyze the marker.

BW Cloning requires the correct bacterial strain is used.

Calculating Cloning Efficiency

orm.htm

Linear DNA fragments electrophoreses through the agarose gel at a rate that is inversely proportional to the log10 of their molecular weight. For convenience, molecular weight is expressed as numbers of base pairs (bps). Use a ruler on your gel or a photograph of your gel to measure the distance each band of DNA has migrated from the sample well, and construct a graph that relates distance migrated to base-pair size of the fragment. Graph a “standard” ( fragments of known molecular weight) on semilog graph paper, and then use the plot to derive the bp size of fragments of unknown size that have run on the same gel side by side with the standard.

HindIII bp size (standard) 23,130 bps Hind III Measure the known sizes first in MM migration. Plot these on a semilog excel spread sheet. Measure the unknown bands MM in migration. Estimate their sizes based on the plot you made.