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

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5. For palindromic restriction enzyme sites, concatamerization of PCR products containing
terminal half-sites reconstitutes the site. For example, end-to-end ligation of DNA
molecules with terminal sequences GGA-3′ and 5′-TCC reconstitutes the BamHI recognition
sequence. This allows the use of shorter PCR primers containing fewer extraneous
nucleotides that do not hybridize to the target sequence.
6. Intermolecular joining of PCR products is stimulated by macromolecular exclusion
molecules, such as PEG 8000, with maximal stimulation occurring in the range 15 to
25% (w/v).
7. Concatamerization and digestion of PCR products containing nucleotides 5′ to the restriction
site generates a small cohesive-ended fragment that can coprecipitate with the fulllength
product and give rise to false positives on ligation into M13. Purification of the
required product by glass bead isolation from low-melting-point agarose before cloning
is therefore recommended. Eighty to ninety percent of clear plaques examined should
then be found to contain the required insert. This figure falls to 10 to 20% if purification
is not performed.
8. To allow analysis of concatamerized PCR products by gel electrophoresis, PEG must first
be removed by extracting with phenol/chloroform because the presence of the polymer
prevents entry of DNA into agarose.
9. When digesting M13 RF DNA with two enzymes that cut at closely spaced sites in the
polylinker, it is preferable to perform the digests sequentially, rather than simultaneously,
even when both enzymes are active in the same buffer, in order to maximize the cutting
efficiency of each enzyme.
10. The number of clear plaques that can be expected depends on the strategy being followed.
Cloning via direct cutting of efficiently recognized terminal restriction sites should yield
50 to 100 clear plaques per transformation. Rather fewer, typically 10 to 20, are produced
by the concatamerization/digestion and blunt-end approaches.
11. M13 clones containing the required insert can quickly be identified by PCR using primers
used for the original amplification. Use sterile toothpicks to transfer phage particles from
individual clear plaques into 0.5-mL microcentrifuge tubes containing all components of
the original PCR mixture minus the template. Vortex lightly and add mineral oil. Heat
to 95°C for 2 min to lyse phage and then perform 25 cycles of the original temperature
regimen. Analyze by agarose gel electrophoresis.
References
1. Buchman, G. W., Schuster, D. M., and Rashtchian, A. (1992) Rapid and efficient cloning of
PCR products using the CloneAmp system. Focus 14, 41– 45.
2. Crowe, J. S., Cooper, H. J., Smith, M. A., Sims, M. J., Parker, D., and Gewert, D. (1991)
Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase
K digestion. Nucleic Acids Res. 19, 184.
3. Krowczynska, A. M. and Henderson, M. B. (1992) Efficient purification of PCR products
using ultrafiltration. Biotechniques 13, 286–289.
4. Liu, Z. and Schwartz, L. M. (1992) An efficient method for blunt-end ligation of PCR
products. Biotechniques 12, 28–30.
5. Bennett, B. L. and Molenaar, A. J. (1994) Cloning of PCR products can be inhibited by Taq
polymerase carryover. BioTechniques 16, 32.
6. Hitti, Y. S. and Bertino, A. M. (1994) Proteinase K and T4 DNA polymerase facilitate the
blunt-end subcloning of PCR products. Biotechniques 16, 802.
7. Clarke, J. M. (1988) Novel non-templated nucleotide addition reactions catalysed by
prokaryotic and eukaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686.