User:Alexander Boehme/Notebook/BioE140L

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AsbF-2 Synthon Construction File

Mix oligios for asbF-2 synthon, do PCA1 protocol (514 bp, pca1)

PCR pca1 with asbF2-NewForward and asbF2-NewReverse by PCA2 protocol (468 bp, pca2)

Digest pca2 with EcoRI/BglII (yes, BglII), gp (452+11+5 bp, pcr_dig)

(get pre-digested vector from JCA of pBca9145-Bca1144 (EcoRI/BamHI, vect_dig)

Ligate pcr_dig and vect_dig, transform, pick white colonies, map, sequence



PCA1 38 uL ddH2O 5 ul 10x expand buffer 5 ul 2mM dNTPs 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) 0.75 ul Expand polymerase Program (can run JCA/PCA1) 2 min initial denature at 94oC 30 sec denature at 94oC 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed] 30 sec extension at 72oC repeat 2-4 30 times total