User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/05

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08/05/10

 * &#x2713; Sequencing order: KAH160-170
 * &#x2713; Order oligos: seq primers for pAct5C/ KFV1
 * &#x2713; Transformation (quick and dirty): pCaSpeR-hs/eGFP-Nintra plasmid (from J. Belote) into T-DH5α

Sequencing order > Confirm that Kozak is at the beginning of constructs --> old vector from insert digest could yield false positive --> Kozak too short to tell difference between old insert vs. new construct --> Forward: P0001
 * 1) KAH160-1 F: good (keep)
 * 2) KAH160-2 F: good
 * 3) KAH161-1 F: good (keep)
 * 4) KAH161-2 F: good
 * 5) KAH162-1 F: good (keep)
 * 6) KAH162-2 F: good
 * 7) KAH163-1 F: good (keep)
 * 8) KAH163-2 F: good
 * 9) KAH164-1 F: good (keep)
 * 10) KAH164-2 F: bad read (non-specific)
 * 11) KAH165-1 F: okay, seq match after manual corrections (keep)
 * 12) KAH165-2 F: bad read (non-specific)
 * 13) KAH166-1 F: fshPcCD, should be flyPcCD; good seq (keep)
 * 14) KAH166-2 F: same
 * 15) KAH167-1 F: flyPcCD, should be fshPcCD; good seq (keep)
 * 16) KAH167-2 F: same
 * 17) KAH168-1 F: good (keep)
 * 18) KAH168-2 F: bad read (poor quality)
 * 19) KAH169-1 F: bad read (poor quality); keep and use based on digest result from 8/04/10
 * 20) KAH169-2 F: bad read (no priming)
 * 21) KAH170-1 F: good (keep)
 * 22) KAH170-2 F: good

Order oligos

> Ian (Depace lab) sequenced off the end of the R. luciferase gene in pAct5C using 5'-ATCGGACCCAGGATTCTTTT. Use this sequence data to design reverse sequencing primers for reading back towards the R. luc insert (the spot where I cloned in the microMCS).
 * 1) pA5Cseq r1; 5'-CGGTTTGGTGTCTCTGGATTAG
 * 2) pA5Cseq r2; 5'-GCAGCAACTTCTTCGTCACAC

> "Nintra" fly deg. tag BB primers and sd mutagenesis primer --> See Neuburger et al. 2006, Genetics for details on eGFP-Nintra
 * 1) BB_Nintra f; 5'-cctttctagaAAGAATAGTGCAATAATGCAAACG
 * 2) BB_Nintra r; 5'-aaggctgcagcggccgctactagtAATGTAGATGGCCTCGGAAC
 * 3) mut_Nintra; 5'-TCGCCGCGATCaAATTCCGATTGGA


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