Interference No. 102,572
to ampicillin. Cabilly ¶ 5 (CR-39).
(11) According to Holmes, six fragments, A-F, had to be isolated to make the heavy
chain expression plasmid, pGammaCEAInt2. To make the first fragment, Holmes
digested pHGH207-1* with AvaI, filled in, digested with BamHI, treated with BAP and
purified the large fragment by PAGE (fragment A). Holmes, ¶ 14. He digested
pGamma11 with PstI, the fragment was purified by PAGE, digested with AvaII, filled in,
and digested with TaqI. The 375 bp fragment was isolated by PAGE (fragment B).
Holmes, ¶ 15. He digested pGamma298 with TaqI, BamHI, and isolated the 496 bp
fragment by PAGE (fragment C). Holmes, ¶ 16. Holmes ligated fragments A, B and C and
transformed the ligation reaction into E. coli. The resultant transformants were analyzed by
restriction digestion to confirm the construction of pGammaCEAInt1. Holmes, ¶17 (CR-
31). Holmes used a 15 nucleotide DNA primer in a primer repair reaction to introduce the
initiation codon into an AluI to RsaI fragment of pGamma 298 (fragment D). Holmes, ¶ 18
(CR-31). He digested pGamma298 with PstI, BamHI, HpaII and purified the fragment by
PAGE (Fragment E). Holmes, ¶ 18 (CR-31). He digested pGammaCEAInt1 with EcoRI,
filled in, and digested with BamHI. The then treated this fragment with BAP and purified
the fragment by PAGE (Fragment F). Holmes, ¶ 18. He then ligated fragments D, E and F
and transformed the ligation reaction into E. coli. The plasmid, pGammaCEAInt2 was
said to be confirmed by restriction analysis and sequencing. Holmes, ¶ 18 (CR-31).
(12) Holmes stated that he prepared the expression plasmid pGammaCEAtrp207-
15
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