Repeat Midipreps to select new clones for sequencing for failed sequences from Oct. 2

From Real Vegan Cheese
Jump to navigation Jump to search

Midipreps & DNA quant. to select new clones for sequencing of failed 02Oct2014 reactions and culture storage


These experiments are continued from Submitting 10 casein and FAM20C in pD1214 plasmids for sequencing 30Sep2014. As the sequences we received October 2, 2014 either failed or were wrong/ambiguous for four of our constructs: bovine alpha S1 (1), bovine alpha S2 (Kex +) (3), human kappa casein (Kex -) and bovine beta A2 (6), we are midiprepping (two, where possible) new clones of each and quantifying the plasmid DNA for each to ready them for sequencing. We will repeat sequencing with primers redesigned to give clearer reads at the 3' ends of our insert regions.

Plasmid midipreps for bovine alpha S1 (1), bovine alpha S2 (Kex +) (3), bovine Beta A2 (6):pD1214

  • Participants: Johan, Aaron, Rebecca, Allen, Lafia, Menakshi (Aaron did the protocol with some help from Johan and Allen)
  • Notes: Rebecca
  • Date: 10/3/2014
  • Location: BioCurious
  • Aims: Midiprep colonies from P.FAKS.bovAlphaS1.S (1), P.FAKS.bovAlphaS2(Kex+).S (3), P.FAKS.bov.Beta(A2).S (6)
    • (We did not get to midiprep P.FAKS.humkappa(kex-).S (5) tonight because we had to duplicate the glycerol stock). ***We should have also made glycerol stocks of bovine alpha S1 (1), bovine alpha S2 (Kex +) (3), bovine Beta A2 (6):pD1214!!
  • Materials and Methods*
    • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎)
      • Changes to protocol: Step 9: centrifuge 5 min the 2nd time, not 1 min
    • Cultures were grown in 6 ml culture with 6 microliters 50mg/ml carbenicillen from Teknova; they had been innoculated on 10/2/2014 with with single colonies from 9/12/2014 transformation plates; plastic loops (not individually wrapped but from a new bag) were used; cultures were incubated for 24 hours.
    • Each was done twice (clone # A and clone # B) to increase the chances of getting the correct sequence because Electra negative control plates had colonies on them, suggesting possible contamination.

Storage of human Kappa casein(Kex-):pD1214 in dH5alpha E. coli(5)

October 3, 2014

  • Participants: Meenakshi, Lafia, Rachel
  • Notes: Rachel
  • Location: BioCurious
  • Aims: The September 12, 2014 transformation plate for human Kappa casein(Kex-):pD1214 to dH5alpha E. coli(5) had only two colonies. Potential issue with toxicity of full human kappa casein protein leakily expressed in E. coli. As we will midiprep plasmid DNA from the second and final colony, we need reserve stocks of this clone in case it contains our desired plasmid. This is especially important given an attempted re-transformation of this plasmid from the September 12, 2014 Electra cloning yielded no colonies.
  • Materials & Methods:
    • We stored this culture in two forms:
      • 1) Combined 150uL of 02Oct2014 O/N culture with 150uL of 50% glycerol in 2.0mL microfuge tube. Placed tube in Ziploc labeled "glycerol stocks casein/FAM20C:pD1214 -> dH5alpha iGEM REal Vegan Cheese."
        • Stored in BioCurious -20°C freezer in iGEM Styrofoam box, as we don't have access to a -80°C. Stock should stay good for ~6 months.
      • 2) Plated 150uL of 02Oct2014 O/N culture to Prewarmed LB agar plates with Carb-50, 100mm (Teknova) using individually wrapped plastic spreader.
        • Incubated this plate at room temperature rather than 37°C to limit protein expression and mitigate potential toxicity.

Plasmid midiprep of human Kappa casein(Kex-):pD1214

October 4, 2014

  • Participants: Johan, Lafia, Meenakshi
  • Notes: Lafia & Maria, transcribed by Rachel
  • Location: BioCurious
  • Aims: Sequencing plasmid DNA extracted from first colony of P.FAKS.humKappa(Kex-).S:pD1214 12Sep2014 transformation into dH5alpha E. coli yielded noisy, incorrect data. That clone unlikely to contain desired insert. Today, we will perform a plasmid midiprep on DNA from the second and final 12Sep2014 transformation colony. After UV spec quantification/purity analysis, this DNA will be submitted for sequencing.
  • Materials & Methods:
    • Midiprep source culture grown for 24 hours in LB carbenicillin liquid media, 37°C shaking incubator. This colony designated "5A."
    • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎) with the following specifics:
      • Followed "Centrifugation Protocol" stream without the Step 1) "alternative" that we usually perform, as our E. coli cultures incubated longer than usual.
        • Centrifugation of 50mL conical tubes took place in a Sorvall Legend T centrifuge.
        • Centrifugation of 2mL kit spin columns and 2.0mL microfuge elution tubes took place in benchtop Beckman microfuge E.
      • Removed 10uL eluted DNA for quantification. Removed 10uL for sequencing. Remaining 130uL DNA stored in Johan's pink box, BioCurious -20°C freezer.

UV spec readings for plasmid DNA quantification & purity, 03 & 04Oct2014 midipreps

October 4, 2014

  • Participants: Lafia, Meenakshi, Johan
  • Notes: Lafia & Maria
  • Location: BioCurious
  • Aims: goal was quantification of the plasmids
  • Materials and Methods: used Beckman Coulter DU 640 Spectrophotometer
  • Results: All concentration readings should be multiplied by 10 to account for 1:10 dilution of plasmid DNA in diH2O prior to UV spec readings.

RVC Spec oct.5 - Sheet1.jpg

  • Discussion, Next Steps: