User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/16

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07/16/10

 * &#x2713; Sequencing order: KHF01-04
 * &#x2713; Oligo order: micro BioBrick cloning site for pAct5C plasmid; micro BB cloning site for TetO-FUW-oct4 lentivirus vector

Sequencing order > fwd = P0001, rev = P0002
 * 1) KHF01 fwd: good
 * 2) KHF01 rev: good
 * 3) KHF02-1 fwd: good
 * 4) KHF02-1 rev: good
 * 5) KHF02-2 fwd: n/d
 * 6) KHF02-2 rev: n/d
 * 7) KHF03-1 fwd: good
 * 8) KHF03-1 rev: good
 * 9) KHF03-2 fwd: n/d
 * 10) KHF03-2 rev: n/d
 * 11) KHF04-1 fwd: mutation at 575 bp (V > A missense mutation)
 * 12) KHF04-1 rev: mutation at 575 bp (V > A missense mutation)
 * 13) KHF04-2 fwd: good
 * 14) KHF04-2 rev: good

Oligo order

> micro MCS for pAct5C --> "micro MCS site for BioBrick inclusion" from V0200: CACCgcggccgc(NotI)AGTGTCAAGGGTactagt(SpeI)C --> Vector: Act5C promoter - SpeI - R. luciferase - NotI --> Replace SpeI/NotI fragment with NotI/SpeI micro MCS; destroy SpeI w/ XabI overhang; destroy NotI site with a mutation before NotI overhang --> Oligos:
 * 1) microMCS 1; 5'-ctagaCACCgcggccgcAGTGTCAAGGGTactagtC
 * 2) microMCS 2; 5'-ggccGactagtACCCTTGACACTgcggccgcGGTG

> micro MCS for TetO-FUW-oct4 lentivirus vector (for Pc-ATF expression in stem cells) --> Replace EcoRI fragment with EcoRI/XbaI micro MCS; destroy second EcoRI site with a mutation before the EcoRI overhang --> Oligos:
 * 1) microMCS2 1; 5'-aattcGAGGCCGCAtctagaTt
 * 2) microMCS2 2; 5'-aattaAtctagaTGCGGCCTCg


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