The PCR technique requires only small amounts of DNA (<1ug) from the parental genotype. In this case 400ng of a single seedling mini-DNA preparation were used as the target DNA in the amplification step. A unique fragment of the bz2-mu1 sequence including the entire Mu element was excised from the clone and replaced by the same amplified region of the Bz2 locus from the progenitor genotype lacking the Mu insertion.
500ng of the unique Eco47111-BglII fragment of Bz2 sequence were produced in two rounds of amplification. The first round primers (1 and 2 above) resulted in amplification of a 338 bp piece of DNA. The set of primers used in the second round of amplification (1 and 3 above) met two criteria: 1) they included the two unique restriction enzyme sites necessary for cloning and 2) at least one of the primers was internal to the fragment amplified in the first round yielding enrichment for the desired product. Buffers and reaction conditions were as suggested by the distributers of the Taq polymerase (Perkin-Elmer/Cetus). The amplification program used is 30X (1' 95 C, 1' 55 C, 2' 74 C) followed by 5X (1' 74 C).
Both the bz2-mu1 clones and the
PCR amplified product were digested with Eco47111 and BglII
for compatible cloning. Digested products were separated in a 1% NuSieve
low melting agarose gel. The 4kb bz2-mu1 clone fragment and the
316 bp amplified genomic Bz2 fragment were each cut out of the gel
with a razor blade. The 1.5kb Mu containing fragment of the bz2-mu1
clone was well separated and was left behind in the gel. Gel samples were
melted at 68 C for 10 min. 5 ul samples of each DNA were mixed and cooled
to 37 C. 10 ul of 2x ligase reaction buffer plus ligase (BRL) were added
and the reaction was allowed to sit at room temperature for 4-20 hours.
The sequence of one of the reconstructed Bz2 clones indicates that
the PCR amplified region of Bz2 is identical to the corresponding
bz2-mu1 sequence except that the Mu1 element and 9bp host
sequence duplication were eliminated.
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