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

asbF2-NewForward ccaaaGAATTCCGTCTCATAGAAACCCATCC

asbF2-NewReverse gactgAGATCTCGTCTCAGGTCTCAGGATCCAG


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

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

Notes

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