선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
제안 방법
- 2 for overview). Following this procedure, it required the picking of 25 to 50 individual clones to identify a positive plasmid, no matter if one or two enzymes were used for the digestion of the BAC clones. The number of clones we had to analyze by PCR amplification to obtain the targeted DNA was lower than expected, if one assumes that all fragments generated by the restriction digests are equally likely to be inserted into the vector (see Table 1).
- To clone a distinct genomic fragment of defined size and sequence, BAC clones containing the targeted DNA were first identified. For this purpose, we performed a BLAST search on the entire genome using cDNA sequences. The result is in most cases, as expected, one supercontig containing the entire cDNA on one chromosome (Fig.
- The “Map View” option reveals exon/intron structure and BAC clones stored at TIGR containing the genomic region corresponding to the employed cDNA. To isolate smaller fragments from the BAC clones, we performed another genome wide BLAST, this time using both BESs as query templates. This revealed their positions in the genome and made it possible to retrieve the sequence of the entire insert.
- For this purpose, we constructed a virtual Bluescript vector containing the presumed sequence of the targeted genomic fragment. With this sequence information, we identified a primer (18-22 bases) on the genomic DNA fragment compatible to the T7 or T3 site of the vector using a commercial primer design program (Oligo 6.8, Molecular Biology Insights, Inc). We picked the bacterial clones, placed them first into the PCR reaction mix and then into bacterial medium using 96 well plates.
- This revealed their positions in the genome and made it possible to retrieve the sequence of the entire insert. With this sequence, we generated a restriction map, which we applied to design a one-step cloning strategy to isolate fragments of 1 to 10kb straight from the BAC DNA. These restriction maps were generated with a commercial software (Gene Construction Kit 2.
대상 데이터
- PCR conditions and restriction digests. The BAC clones were all purchased for a nominal fee from the Children’s Hospital Oakland Research Institute (http://bacpac.chori.org). All used restriction enzymes are commercially available.
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