John M. S. Bartlett.pdf - Bio-Nica.info

496 Preston
MgCl 2 concentrations between 0.7 and 1.0 mM gave the best results (17,18); however,
MgCl 2 concentrations between 0.5 and 5.0 mM have been reported.
3. Organic solvents and ethidium bromide are hazardous materials. Always handle with
tremendous caution, wearing gloves and eye protection. Contact your hazardous waste
department for proper disposal procedures in your area.
4. Ampicillin-resistant bacteria secrete β-lactamase into the media, which rapidly inactivates
the antibiotic in regions surrounding the growing bacterial colony. Thus, when bacteria are
growing at a high density or for long periods (>16 h), ampicillin-sensitive satellite colonies
will appear around the primary colonies (which are white in blue-white selections).
This problem can be ameliorated (but not eliminated) by substitution of carbenicillin for
ampicillin on agar plates.
5. When designing primers with restriction enzyme sites and 5′-overhangs, note that the
5′-overhang should not contain sequences complementary to the sequence just 3′ of the
restriction site because this would facilitate the production of primer-dimers. Consider
the primer 5′-ggg.agatct.CCCAGCTAGCTAGCT-3′, which has a XbaI site proceeded by
a 5′-ggg and followed by a CCC-3′. These 12 nucleotides on the 5′-end are palindromic
and can therefore easily dimerize with another like primer. A better 5′-overhang would
be 5′-cac.
6. When cloning a gene from a recombinant library by PCR, remember that not all genes are
created equally. Genes with high GC contents have proven more difficult to clone than
most. Several researchers have made contributions in a search for factors to enhance the
specificity of PCRs. Nonionic detergents, such a Nonident P-40, can be incorporated in
rapid sample preparations for PCR analysis without significantly affecting Taq polymerase
activity (19). In some cases, such detergents are absolutely required to reproducibly detect
a specific product (20) presumably because of inter- and intrastrand secondary structure.
Tetramethylammonium chloride has been shown to enhance the specificity of PCRs by
reducing nonspecific priming events (21). Commercially available PCR enhancers are
also available.
7. A critical parameter when attempting to clone by PCR is the selection of a primer annealing
temperature. This is especially true when using degenerate primers. The primer melting
temperature (T m ) is calculated by adding 2° for AT base pairs, 3° for GC base pairs; 2°
for NN base pairs, and 1° for IN base pairs. Most PCR chapters suggest you calculate the
T m and set the primer annealing temperature to 5 to 10°C below the lowest T m . Distantly
related gene superfamily members have been cloned using this rationale (22). However, I
have found that higher annealing temperatures are helpful in reducing nonspecific priming,
which can significantly affect reactions containing degenerate primers.
Acknowledgments
I thank my colleagues, especially Peter Agre and William B. Guggino, for their
support and helpful discussions. This work was supported in part by NIH grants
HL33991 and HL48268 to Peter Agre.
References
1. Reizer, J., Reizer, A., and Saier, M. H., Jr. (1993) The MIP family of integral membrane
channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathways
of evolution, and proposed functional differentiation of the two repeated halves of the
proteins. Crit. Rev. Biochem. Mol. Biol. 28, 235–257.
2. Knepper, M. A. (1994) The aquaporin family of molecular water channels. Proc. Natl.
Acad. Sci. USA 91, 6255–6258.