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

The Vectorette Method 397
Fig. 2. Three vectorette “libraries” were created using the blunt-ended restriction enzymes:
PvuII (lanes 1 and 2), StuI (lanes 3 and 4), and RsaI (lanes 5 and 6). PCR was performed using
oligos specific for the centric arm of the pYAC4, 1089, and the universal vectorette oligo, 224.
In A, 5 µL of Perfect Match have been added to each PCR, whereas in B, this has been omitted.
Ten microliters of untreated product were loaded on a 2.5% agarose minigel in lanes 1, 3, and
5, whereas samples in lanes 2, 4, and 6 were first digested with EcoRI to release the vector
arm from the genomic fragment. Lane 7 contains HaeIII fragments of ΦX RF DNA (Gibco
BRL, Paisley, Scotland). StuI-digested YAC has failed to produce a PCR product (A, lanes 3 and
4), probably through the lack of an enzyme site close to the vector/insert junction. PvuII- and
RsaI-digested YAC yields products of approximately 800 and 500 bp (lanes 1 and 5), respectively,
which on digestion with EcoRI release vector fragments (V) of the predicted size 287 bp together
with the terminal PvuII and RsaI fragments of the YAC insert (500 and 200 bp).
7. When the reaction mix is equilibrated, add 1 µL of T4 DNA ligase (1 U/µL) and incubate
at 37°C. After 1 h, add 400 µL of 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, and mix
thoroughly. The vectorette library may now be stored in aliquots at –20°C.
8. Two sets of PCR mixes need to be prepared for each vectorette library constructed. The
first contains a primer, 1091, specific for the “right” arm of the YAC vector (i.e., the
acentric arm encoding the URA3 gene) together with the vectorette-specific oligo (224),
whereas the second contains a primer, 1089, specific for the “left” arm of the YAC vector
(i.e., the centric arm, which contains the CEN4 gene) together with 224 (see Table 1).
Each reaction is carried out in 50 µL buffer described in Subheading 2., item 3, including
5 µL of Perfect Match (see Note 3 and Fig. 2) using the following cycling conditions:
94°C for 1 min, 1 cycle, followed by 93°C for 1 min, 65°C for 1 min, and 72°C for 3 min,
38 cycles, and followed by 72°C for 5 min, 1 cycle. For IRP, see Note 4 for suggested
primer sequences.
9. Confirmation that PCR products originate from the terminal sequences of YAC clones can
be obtained by demonstrating the presence of the YAC vector cloning site in the hybrid
fragment. This is done by digesting the PCR product with a restriction enzyme that cleaves
within the cloning site. To 9 µL of PCR product, add 1 µL of 10× restriction enzyme
buffer (see manufacturer’s recommendation), 10 U of enzyme, and incubate for 1 h at
37°C. When the vector is pYAC4, 10 U of EcoRI may be added directly to 9 µL of PCR
product, without addition of enzyme buffer. Restriction fragments can be visualized on a
2.5% agarose minigel containing ethidium bromide (0.5 µg/mL; Fig. 2). Note: ethidium
bromide is a powerful mutagen and gloves should be worn at all times. The distances
from the primer sequences described in Subheading 2., item 3 to the EcoRI cloning site
of pYAC4 are given in Table 2.