Biology Reference
In-Depth Information
5. Clean the PCR reaction using the QIAquick PCR Purification Kit (Qiagen) to
remove primers and polymerase, and prepare the DNA fragment for endonu-
clease digestion.
6. Analyze part of the PCR reaction product by RFLP (or sequencing) (
see
Note
7
) and include restriction enzymes that are used in the primer tags to ensure that
there are no internal sites.
7. Digest the fragment with restriction endonucleases corresponding to the sites
used in the primer tags.
8. Clean the reaction product to remove the enzymes used for the restriction
digests with the QIAquick PCR Purification Kit (Qiagen). Gel purification of
the fragment following digestion is not necessary if only one band is produced.
If multiple bands are produced, then gel purification of the fragment
is
required
for a specific cloning strategy. Use BlueView stain rather than ethidium bromide
(EtBr) for visualization and isolation of DNA fragments (
see
Note 5
).
9. Ligate the target locus with T4 DNA ligase to the restricted and cleaned pOK12
amplicon overnight at 16°C.
10. Use a portion of the ligation to transform OneShot Top10
E. coli
per the manufac-
turer's instructions (Invitrogen), and select clones on LB plates supplemented
with 50 μg/mL kanamycin sulfate at 37°C.
11. Analyze kanamycin-resistant clones by PCR, RFLP, and sequence analysis for
pOK target locus.
12. Store cultures indefinitely in 20% glycerol at -80°C, or for no more than a
couple of weeks at 4°C. The plasmid containing the
vtp
target locus is termed
pOKvtp (
see
Note 6
and
Fig. 1
.)
3.1.2. Construction of Inactivation and Reconstitution Plasmids
1. Design inverted endonuclease-tagged primer sets specific to the pOK-
target locus
such that PCR will generate an amplicon of the entire pOK-
target locus
plasmid,
with a
deletion
where the resistance cassette is intended to be placed. For the
inactivation construct
, a major intragenic portion of the target gene is deleted
and replaced with the
flgB
-promoted kan cassette, such that a double-crossover
homologous recombination event will result in allelic replacement of the target
gene with the resistance cassette (i.e.,
flgB-kan
)(
see
Note 8
for construction
details relevant to the
vtp
inactivation plasmid, pOKvtpKO, and
Fig. 1
for an
illustration of this plasmid). For the
reconstitution construct
, this
deletion
should
be intergenic, where a double-crossover homologous recombination event will
result in the insertion of a resistance cassette (i.e.,
flaB-gent
) at a nearby location
(
see
Note 9
for construction details relevant to the
vtp
reconstitution plasmid,
pOKvtpRECON, and
Fig. 1
for an illustration of this plasmid). Construction of
the inactivation and reconstitution plasmids is achieved by inverse-PCR-mediated
cloning utilizing the pOK-
target locus
plasmid and the resistance cassettes in
pTABhFlgB-Kan and pTABhFlaB-Gent. Primers should include restriction tags
that are complementary to the restriction endonuclease sites flanking the particular
Search WWH ::
Custom Search