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Permits
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These permits may be required for shipping this product:
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Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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Vector Information
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Size (kb): 9.8199996948242190 Vector: pDO184 (cosmid) Construction: pBR322, pK184 Marker(s):ampR,kanR Construct size (kb): 9.819999694824219 Features: insert detection: lacZ'
marker(s): ampR
marker(s): kanR
other: lambda cos site
replicon: p15A
replicon: pMB1
MCS: EcoRI...HindIII
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Applications
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vector containing primer sites useful for sequencing vector for generating nested deletions of cloned inserts vector permitting visual detection of recombinants
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Comments
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Restriction digests of the clone give the following sizes (kb): EcoRI--10.0; HindIII--10.0; XbaI--9.2, 1.0; BglI--2.8, 2.35 (doublet), 1.55, 0.75, 0.28; PstI--4.0, 3.7, 2.5. Plasmid contains the following restriction sites (approximate kb from nt 1): BamHI--4.42, 4.77; BglI--1.96, 4.60, 5.32, 5.56, 7.88, 9.42; EcoRI--4.40; HindIII--4.45; PstI--0.65, 4.44, 8.00; PvuI--0.97, 4.57, 8.13; SmaI--4.41; XbaI--3.45, 4.42. A series of overlapping deletion segments can be generated by partial digestion with a restriction enzyme not found in the vector, followed by complete digestion with lambda terminase. Resulting fragments are blunted and circularized by ligation, and transformants are selected for either ampicillin or kanamycin resistance. The following restriction sites do not cut pDO17: Acc65I AflII ApaI AscI AvrII Ecl136II KpnI NotI PacI PmeI PmlI SacI SfiI SnaBI SpeI SrfI Sse8387I SwaI XbaI. IMPORTANT: To prevent amplification of a rearranged and/or deleted cosmid, we recommend streaking on LB + amp plates at 30C and picking small colonies for liquid culture. Cosmid vector containing two compatible origins of replication, for generating bidirectional deletions of large inserts to facilitate sequencing. AmpR transformants can be sequenced in the clockwise direction using the cosL primer 5'-TCATAAATAGCGAAAACC-3'. KanR transformants can be sequenced in the counterclockwise direction using the cosR primer 5'-ACTTTACGGGTCCTTTCCG-3'.
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Media
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ATCC Medium 1065 (see below) plus ampicillin (50.0 mcg/ml) plus kanamycin (20.0 mcg/ml)
ATCC Medium 1065:
Tryptone (Difco 0123), 10.0 g
Yeast Extract (Difco 0127), 5.0 g
NaCl, 10.0 g
Distilled water, 1.0 L
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References
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Ahmed A. Double-origin vectors for isolating bidirectional deletions useful in DNA sequence analysis. Gene 141: 71-73, 1994. PubMed: 8163177
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