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SubmittingPlasmidSequencingOct
From 2014.igem.org
October 10, 2014: DNA pick-up by [http://sequetech.com/ 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
- Aims: Confirm correct DNA sequence of our casein inserts in pD1214 vector, including sequencing duplicate, new clones for 4 wrong/failed/ambiguous sequences from Submitting 10 casein and FAM20C in pD1214 plasmids for sequencing 30Sep2014, "Results and Discussion" October 1, 2014 and new human Alpha casein S1 construct.
- Sequencing Strategy & Methods:
- Details about samples submitted for sequencing in table, below.
- Submitting 10uL aliquots of 7 samples:
- 3 constructs submitted in duplicate -- plasmid preps performed on two separate E. coli clones:
- P.FAKS.bovAlphaS1.S:pD1214 (clones 1A, 1B), P.FAKSbovAlphaS2 Kex (+):pD1214 (clones 3A, 3B), and P.FAKS.bovBetaA2.S:pD1214 (clones 6A, 6B) as sequences were wrong/ambiguous the 30Sep2014 sequencing linked above. (see Repeat Midipreps to select new clones for sequencing for failed sequences from Oct. 2)
- P.FAKS.humKappa(Kex-).S:pD1214 (clone 5A) also repeat of failed sequencing 30Sep2014, but this is a prep of only remaining transformation colony. (see Repeat Midipreps to select new clones for sequencing for failed sequences from Oct. 2
- 3 constructs submitted in duplicate -- plasmid preps performed on two separate E. coli clones:
- 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 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). CYC1 R 182: 5' TACACGCGTCTGTAC 3' 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:
October 13, 2014
- 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.
