Submitting 4 repeat and 1 new casein in pD1214 plasmid for sequencing 10Oct2014

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October 10, 2014: DNA pick-up by Sequetech to sequence 8 samples, detailed below

  • Location: BioCurious
  • Participants: Rachel (set up sample pick-up), Emi (gave samples to Sequetech rep), Craig (sequence analysis)
  • Notes: Rachel
  • Sequencing Strategy & Methods:
    • Details about samples submitted for sequencing in table, below.
    • Submitting 10uL aliquots of 7 samples:
    • Submitted 15uL of 8th sample: P.FAKS.humAlphaS1.S:pD1214 ("HA") Cloning human alpha casein S1, 04Oct2014 and on.) Larger vol., as not quantified by UV spec.
    • Sequetech rep. collected samples at ~3:00pm today.
    • All to be sequenced in three reactions: one forward (with primer TEF-264-FW) and two reverse (with primers PMYR3 and CYC1 R 182).
      • TEF-264-FW is a custom primer synthesized through Sequetech: primer sequence recommended by DNA 2.0, suppliers of pD1214. This primer will be maintained in-house for future sequencing reactions. (Note change from sequencing described August 6, 2014, as previous forward primer, Alpha-factor 146-forward, anneals to the FAKS sequence, now inside our redesigned DNA inserts.)
      • PMYR3 is an in-house Sequetech primer.
      • CYC1 R 182 is a custom primer synthesized through Sequetech. We designed this primer 09Oct2014 to anneal downstream of PMYR3 in the CYC1 terminator region, as PMYR3 may be too close to 3' end of our plasmid insert to give quality sequence covering 3' end. Primer will be maintained at Sequetech for future reactions.
        • Requesting reactions with both reverse primers to compare efficacy of PMYR3 and CYC1 R 182.
Primer Sequences 
 Binds to TEF promoter and reads through in the 5'-3' direction (N-terminus of signal peptide).
 Binds to the terminator (CYC1) and reads through in the 3'-5' direction (C-terminus of casein).

 Binds to the terminator (CYC1) downstream of PMYR3 and reads through in the 3'-5' direction (C-terminus of casein).

Details for 24 sequencing reactions on plasmid DNA from 8 clones:

Sample details from Sequetech sequence pick-up, 10Oct2014.png

October 13, 2014

Sequencing data summary 10-11-14.png

      • All clones in this table (with the exception of hKappaKex-) were sequenced with PMYR3, TEF FW, and CYC1 R 182 providing reliable multi-directional read throughs of each construct.