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

T4 DNA Polymerase 473
5. Room temperature (25°C) was chosen for the reaction since T4 DNA polymerase has
excessive exonuclease activity at 37°C.
6. The T4 polynucleotide kinase works well at room temperature as opposed to the higher
reaction temperature (37°C) regularly used (8).
7. Although the PCR products purified directly by alcohol precipitation after end repairing
are sufficient for routine cloning, passing the repaired PCR product mixtures through a
gel filtration column prior to the alcohol precipitation can greatly enhance the cloning
efficiency.
8. In the ligation reaction, we routinely used 11 molar ratio between vector (dephosphorylated)
and insert. Generally more than 200 recombinant clones can be obtained with 0.4 µL
of the ligation reaction. Therefore, a single ligation reaction for each PCR product is
sufficient for most applications.
9. Because of its reliability and high transformation efficiency, commercial CaCl 2 -treated
competent cells are used for the transformation step. The bacteria strain we routinely used
is DH10B (BRL; MAX efficiency DH10B competent cells). However, various competent
cells can be purchased from companies, such as Stratagene and Invitrogen.
10. Prepacked Sephacryl S-400HR spin columns (MicroSpin S-400HR) can be purchased
from Pharmacia. Alternatively, the spin columns can be prepared from bulk gel filtration
matrix (Sephacryl S-400HR, Pharmacia) as described in other protocol books (8). The
filtration medium (prepacked or bulk filtration matrix) contains 20% alcohol; therefore,
the spin columns should be washed and equilibrated with TE.
References
1. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., et al. (1985)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science 230, 1350–1354.
2. Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by
procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686.
3. Scharf, S. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press,
San Diego, CA.
4. Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA
containing terminal restriction endonuclease recognition sites. Nucleic Acids Res. 18,
6156.
5. Mead, D. A., Pey, N. K., Herrnstadt, C., Marcil, R. A., and Smith, L. M. (1991) A universal
method for the direct cloning of PCR amplified nucleic acid. Bio/Technology 9, 657.
6. Kovalic, D., Kwak, J., and Weisblum, B. (1991) General method for direct cloning of DNA
fragments generated by the polymerase chain reaction. Nucleic Acids Res. 19, 4560.
7. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. (1991) Construction of T-vectors,
a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids
Res. 19, 1154.
8. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
9. Wang, K., Koop, B. F., and Hood, L. (1994) A simple method using T4 DNA polymerase to
clone polymerase chain reaction products. Biotechniques 17, 236–238.
10. Heery, D. M., Gannon, F., and Powell, R. (1990) A simple method for subcloning DNA
fragments from gel slices. Trends Genet. 6, 173.