User:Alexander Boehme/Notebook/BioE140L

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Line 36: Line 36:
PCA1
PCA1
 +
38 uL ddH2O
38 uL ddH2O
 +
5 ul 10x expand buffer
5 ul 10x expand buffer
 +
5 ul 2mM dNTPs
5 ul 2mM dNTPs
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)

Revision as of 13:21, 12 March 2013

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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

Notes

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