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

522 Jones and Winistorfer
The yield of colonies from plate A is >2 times that from plate B + C, confirming a high
percentage of recombinants in plate A. Plate D is a transformation control, and should
yield a thick lawn of colonies. Plate E is an antibiotic control, and should yield no colonies
since the bacteria that have not been transformed are sensitive to ampicillin. Only 25 µL
of cells are used for the control plates D and plate E, so that only one BRL tube, which
contains 200 µL of bacteria, needs to be used.
7. Screen plasmids by placing individual colonies in 2 mL of LB broth containing 100 µg/mL
of ampicillin. Grow the colonies at 37°C for 6–24 h.
8. Screen the plasmids by removing 2 µL of the LB broth, place it directly in a PCR tube,
and amplify for 25 cycles (see steps 2–4) using primers that flank the mutated site or
insert (e.g., M13 primers) (16).
9. In a mutagenesis protocol, a base pair can be mutated to either create or remove a restriction
endonuclease site. In particular, the degenerate amino acid code allows one to create
or eliminate a restriction endonuclease recognition site without altering the amino acid
encoded at that site. Screen for the mutation by adding 3 U of the restriction endonuclease
and 1 µL of the appropriate 10× restriction buffer directly to 5 µL of the unpurified PCR
product in a total volume of 10 µL.
10. Assess cutting by minigel analysis. Typically, 50 to 100% of the clones contain the
recombinant of interest. Then, purify the plasmid and sequence the mutated region (see
Note 7).
4. Notes
1. Other investigators have used Pfu DNA polymerase instead of Taq DNA polymerase
in recombination PCR (6). Pfu DNA polymerase has better fidelity than Taq DNA
polymerase (17).
2. The primers that introduce point mutations (primers 1–4 in Fig. 2 and primers 1 and 3 in
Fig. 3) are designed to generate 15to 45 bp of homology between each end of one PCR
product relative to the other PCR product. In all recombination PCR protocols, 24 bp of
homology works very well, and alterations that generate long regions of homology do
not work noticeably better. Decreasing the length of homology from 25 to 12 bp in an
early protocol did decrease the transformation efficiency four- to five-fold. Single point
mismatches lie no closer than six nucleotides from the 3′ end of a primer and are frequently
placed toward the middle. Placing point mutations near the 5′ end of each mutating primer
will generate two PCR products whose mutated ends have >24-bp of homology. Multiple
point mismatches should be placed in the middle or toward the 5′ end of a primer, with
primer lengths long enough to create 24-bp of homology between the mutated ends of the
two PCR products. Primers that are nonmutating are generally 24 to 30 nucleotides long.
These nonmutating primers can be designed to anneal to the β-lactamase gene so that they
can be used with a variety of different plasmids. Frequently, the mutating and nonmutating
primers are designed to be perfect complements to each other.
For site-directed mutagenesis, since unique restriction endonuclease recognition sites
almost always bracket the insert, the same linearized templates can be used for the
mutagenesis of any single site in the insert. Primers 2 and 4 are nonmutating (see Fig. 3),
and are conserved for each new site targeted for mutagenesis, so that only two new
primers need to be generated for each site targeted for mutagenesis (via primers 1 and 3).
Furthermore, only approx one half of the length of the entire template needs to be amplified
in each of the two PCR amplifications, facilitating the mutagenesis of large constructs and
permitting considerable flexibility in the primer design and sequence. Recombination PCR
has been used to mutate constructs up to 7.1 kb (18).