User:Alexander Boehme/Notebook/BioE140L

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(Project Description/Abstract)
(Notes)
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* Place some notes here for visitors
* Place some notes here for visitors
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* Example: This project is currently on hold until further notice.
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38 uL ddH2O
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5 ul 10x expand buffer
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5 ul 2mM dNTPs
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1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
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0.75 ul Expand polymerase
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Program (can run JCA/PCA1)
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2 min initial denature at 94oC
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30 sec denature at 94oC
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30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
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30 sec extension at 72oC
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repeat 2-4 30 times total
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Revision as of 13:10, 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

Notes

  • Place some notes here for visitors

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

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