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From 2014.igem.org

September 30, 2014: DNA pick-up by [http://sequetech.com/ Sequetech] to sequence 16 samples, detailed below

• Location: BioCurious

• Participants: Rachel (set up sample pick-up), Emi (gave samples to Sequetech rep)

• Aims: Confirm correct DNA sequence of our casein and FAM20C inserts in pD1214 vector. Trouble shoot Electra cloning negative control positive colonies (See Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014, "Results and Discussion" September 28, 2014.) Confirm identity of unclearly labeled DNA, prob. hKcasein Kex+ in pD1214.

• Sequencing Strategy & Methods:
  • Submitting 10uL aliquots of 16 samples, detailed in table below, including 10 new casein and FAM20C in pD1214 constructs.
    • 4 samples submitted in duplicate -- plasmid preps performed on two separate E. coli clones:
      • P.FAKS.humBetaS:pD1214, as previously problematic construct with few transformed colonies (See Troubleshooting failed 12Sep2014 transformation of P.FAKS.humBeta.S for details.)
      • P.FAKSbovBetaB:pD121, FAM20C Kex(+):pD1214 and FAM20C Kex(-):pD1214, as plasmid inserts may not be what we expect. (See Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014, "Results and Discussion" September 28, 2014.)
    • NOTE: DNA concentration for bovAlphaS2 Kex(+) very low. May have to repeat plasmid prep.
  • Sequetech rep. collected samples at ~2:00pm today.
  • All except hkk+R to be sequenced in two reactions: the forward (with primer TEF-264-FW) and reverse (with primer PMYR3) directions. Sample hkk+R sampled in single direction as was likely the sample already sequenced in both directions, September 5, 2014.
    • 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.) See for primer map.
    • PMYR3 is an in-house Sequetech primer.

Primer Sequences 
TEF-264-FW: 5’ TCGATGACCTCCCATTGA 3’
 Binds to TEF promoter and reads through in the 5'-3' direction (N-terminus of signal peptide).
 
PMYR3: 5' CTTCCTTTTCGGTTAGAG 3' 
 Binds to the terminator (CYC1) and reads through in the 3'-5' direction (C-terminus of casein).

Location of sequencing primers indicated by small purple arrows on map of pD1214:

Details for 31 sequencing reactions on 16 samples:

Sequencing Analysis Summary Table (October 1, 2014)

Clone 9A is also poor. Need to remove from stocks.

• Discussion: Of the 11 sequencing reactions we were able to evaluate (excluding all FAM20C, as insufficient length, and Electra cloning negative, as didn't align), there were confirmed issues with 3/11 (27.3%): incorrect sequences for bovine alpha S2 Kex+ (#3) & human beta (#4.2) and what looks like poor template DNA for human kappa Kex- (#5). Given that rate, suggest prepping and submitting two separate clones for each construct in the future.