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




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

Lane 5 is ASFB-2 2013_03_13-JCA-gel1.jpg

Zymo CleanUp Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. Transfer into the Zymo column (small clear guys) spin through, discard waste. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) spin through, discard waste. Add 200 uL of Zymo Wash Buffer spin through, discard waste. spin for 90 seconds, full speed to dry. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction


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