User:Karmella Haynes/Notebook/BioBrick cloning/2010/05/18

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05/18/10

 * &#x2713; Make MV7 (new vector): delete CMV promoter from pcDNA3.1+ neo
 * &#x2713; Make MV3 (new vector, replace BART18): replace neo in pcDNA3.1+ with hygromycin (order reagents)

Make MV7 (pcDNA3.1+ neo ΔpCMV) > Use MV1 (pcDNA3.1+ neo) > Digest w/ SpeI to get rid of CMV promoter

> Digest (Fermentas FD)

> Measure conc.'s

> Ligations

--> R.T./ ~10 min.; Add 50 μL Turbo DH5α (T-DH5α)

Make MV3 (pcDNA3.1+ hygro) > Strategy
 * Clone pcDNA3.1+ neo in Dam-, Dcm- strain (C2925, NEB) to get rid of CpG methylation (blocks BsaBI, BstBI)
 * Cut Neomycin R gene from pcDNA3.1+ neo w/ BsaBI, BstBI (order enzymes)
 * PCR insert: Hygromycin R gene from V0200 (order oligos):
 * 1) Hygro f1 5'-gatc gatgaggatc atg AAAAAGCCTGAACTCACCGC (has BsaBI and Met)
 * 2) Hygro r1 5'-gatc ttcgaa CTATTCCTTTGCCCTCGGAC (has BstBI)


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