F tools of genetic engineering

Editor's Notes

• #8: For instance if you are cloning a piece of DNA that is flanked on the 5´ and 3´ ends by an AvaI site, you could excise it from the source using AvaI. This would give you a fragment with overhangs at the 5´ and 3´ sites that you could then insert into the Ava I site of the plasmid shown here. If the original size of the uncut plasmid was 3.0kb, and you add a 0.75kb insert, you will be able to verify the addition of the insert by analyzing uncut plasmid, cut plasmid, uncut plasmid with insert, and cut plasmid with insert on an agarose gel. Alternatively, if you are cloning a piece of DNA that is flanked at the 5´ end by an XbaI site and at the 3´ end by a KpnI site, you could cut out your DNA of interest using those two enzymes, digest the plasmid vector with the same to enzymes and clone your DNA of interest between those to RE sites of the multiple cloning region. The advantage of this strategy is that your insert will be cloned directionally (i.e., it will be inserted with the 5´ end at the XbaI site of the vector and the 3´ end at the KpnI site). You will be able to predict the orientation of your insert within the vector. (Teaching suggestion: have your students sketch what such a cloning strategy would look like.)
• #9: The Restriction Enzymes Resource on the Promega Website describes in detail the factors that must be considered when performing a restriction digest and gives several examples of typical restriction enzyme reactions. This guide is available at: http://www.promega.com/guides/re_guide/toc.htm
• #10: Promega offers a variety of systems for isolating DNA from agarose gels. These systems can be found here: http://www.promega.com/applications/dna_rna/fragment.htm
• #13: There are two primary methods for transforming bacterial cells: heat shock and electroporation. In both cases, the bacterial cells have to be made competent or permeable to plasmids that you would like the cell to propagate. To create competent cells for either transformation method used, bacterial cells are grown to logarithmic phase and harvested. Cells growing exponentially can be rendered competent more easily than cells at other stages of growth. After harvesting, the cells are treated differently. Chemically competent cells are created using a series of cold salt washes to disrupt the cell membranes, preparing the cells to accept plasmid DNA (1,2). For electrocompetent cells, the cells are chilled and washed with cold deionized water and 10% glycerol (3,4). A low-salt environment is important when electrical currents are involved. To introduce the desired plasmid into chemically competent cells, the plasmid DNA is mixed with chilled cells and incubated on ice to allow the plasmid to come into close contact with the cells. The plasmid-cell mixture then is briefly heated to 45–50°C, allowing the DNA to enter the cell through the disrupted membrane. The heated mixture is then placed back on ice to retain the plasmids inside the bacteria. Many cells do not survive the rapid temperature change but enough maintain integrity to keep the plasmid and, when medium is added, recover and divide. For electroporation, the competent cells also sit on ice with the plasmid DNA. However, the plasmid-cell mixture is exposed to an electrical current, opening pores in the cell membrane so that the plasmid can enter the cell. Some cells do not survive this treatment but many are able to replicate once medium is added. If the plasmid DNA solution has too much salt in it, arcing can occur, compromising the transformation. Depending on the transformation method used, a plasmid can enter the cell through holes or pores in the bacterial cell wall created by salt washes and heat treatment or no-salt washes and electroporation. Both methods allow efficient recovery of transformed cells using antibiotic selection for the plasmid of interest. 1. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–80. 2. Hanahan, D. (1985) In: DNA Cloning: A Practical Approach Vol. 1, Glover, D. M. ed., IRL Press, Oxford, p. 109–35. 3. Calvin, N.M. and Hanawalt, P.C. (1988) High-efficiency transformation of bacterial cells by electroporation. J. Bacteriol. 170, 2796–801. 4. Seidman, C.E. et al. (1994) In: Current Protocols in Molecular Biology, Ausebel, F.M. et al. John Wiley & Sons, Inc., New York, Unit 1.8.