Troubleshooting failed 12Sep2014 transformation of P.FAKS.humBeta.S

Troubleshooting failed transformation of P.FAKS.humBeta.S:pD1214 into dH5alpha E. coli

Overview

Of the September 12, 2014 transformations (gBlocks:pD1214 to the dH5alpha strain of E. coli & controls, above), only one failed: P.FAKS.humBeta.S:pD1214 (Experiments detailed in Cloning 7 casein gBlocks, 12Sep2014 and on.) The following series of experiments shows troubleshooting and successful completion of the transformation.

Repeating transformation of P.FAKS.humBeta.S:pD1214 into E. coli

September 14, 2014

• Aims: Possible that the Electra cloning reaction (12Sep2014) worked, but the transformation reaction failed for this gene. So we are repeating this transformation using the Electra cloning reaction for P.FAKS.humBeta.S:pD1214 from September 12, 2014. We will transform into Mix & Go competent E. coli, strain dH5alpha.

• Participants: Johan, Patrik, Aaron, Meenakshi

• Location: BioCurious

• Materials and Methods:
  • Repeated the transformation using Media:Zymo_E._coli_competent_cells_transformation_protocol.pdf‎ as described in Methods September 14, 2014, above, with the following changes:
    • To attempt to improve transformation efficiency, we added an outgrowth step after 10 minute incubation on ice. Incubated transformation reactions in liquid SOC media: 2.5 hours at 37°C.

September 15, 2014

• Results: No growth for P.FAKS.humBeta.S:pD1214 to dH5alpha E. coli observed on LB carb plates incubated overnight at 37°C.

• Discussion: Second unsuccessful transformation could have been due to failed 1) transformation 2) Electra cloning or 3) resuspension of IDT gBlock DNA. Ideal to quantify P.FAKS.humBeta.S IDT gBlock resuspension to confirm contains DNA, however, we don't have access to a nanodrop UV spec, and quantifying on BioCurious's Beckman Coulter DU 640 spectrophotometer likely to require more DNA than would be prudent to use (see DNA quantification on September 1, 2014: CLONE 082514 humKappaCasein(Kex+).) Failure of Electra cloning possible due to large number of experimental participants and multiple pipetting steps.

• Future Directions: We will repeat both Electra cloning and transformation for P.FAKS.humBeta.S.

Repeating Electra cloning and transformation for P.FAKS.humBeta.S

September 15, 2014

• Participants: Nikola, Johan, Meenakshi, Lafia, Wes, Richard, Matt

• Location: BioCurious

• Aims: As we had two previous unsuccessful transformations (12Sep1014, 14Sep2014) using P.FAKS.humBeta.S:pD1214 cloning reaction from 12Sep2014, we are going to repeat the Electra cloning reaction for this strain, then repeat the transformation.

• Materials & Methods: cloning P.FAKS.humBeta.S into pD1214
  • Used DNA 2.0 Electra cloning kit protocol.

Reaction:

1uL pD1214 (20ng)
1uL (20ng) P.FAKS.humBeta.S or (+) control DNA from Electra cloning kit
2uL Electra Buffer
1uL of Electra enzyme mix
15uL of Water
----
20uL, Room Temperature, 20min.

• • 5uL reaction products used immediate for transformation into E. coli, below. Remainder stored at -20°C, BioCurious freezer. Each tube labeled with date and casein construct ID.

• Materials & Methods, Transforming Mix & Go Competent dH5alpha E. coli with gBlocks in pD1214, above:
  • We used the Zymo Research instruction manual for "Premade Mix & Go Competent E. coli Cells." Media:Zymo_E._coli_competent_cells_transformation_protocol.pdf‎
  • We completed 4 reactions: P.FAKS.humBeta.S:pD1214, one Electra cloning positive control, one pGLO positive control, one cells only negative control.
    • Thawed individual tubes of Mix & Go chemically competent, dH5alpha E. coli cells slowly on ice.
    • Prewarmed LB agar plates with Carb-50, 100mm (Teknova), at 37°C.
    • Added 5uL of each casein:pD1214 or appropriate control to individual thawed tube of 100uL competent cells. Mixed gently.
    • Immediately incubated on ice for 10 min.
    • Plated 100uL of each reaction to LB carb plates, using one individualy wrapped spreader/reaction.
      • Inducible promoter for pGLO positive control requires arabinose, so 200uL of arabinose spread onto that LB carb plate prior to transformation mix being spread.
    • Incubated plates overnight at 37°C.

• Results & Discussion:
  • This transformation reaction yielded colonies, so likely previous failed transformations due to unsuccessful Electra cloning reaction of P.FAKS.humBeta.S on 12Sep2014. We will choose two colonies for plasmid DNA extraction.

Midiprep of P.FAKS.humBeta.S:pD1214 from dH5alpha E. coli

September 17, 2014

• Participants: Patrik, Meenakshi, Lafia, Nikola, Johan

• Location: BioCurious

• Aims: Midiprep TWO colonies of repeat P.FAKS.humBeta.S:pD1214 in dH5alpha. Selecting two colonies as this gene insert has been difficult in the past.

• Materials and Methods:
  • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎):
    • Followed "Centrifugation Protocol" stream with the following specific changes to step 1):
      • In morning, inoculated two colonies from transformation September 15, 2014 each into 12 mL LB Amp liquid media. Incubated in 37°C shaking incubator until 7pm.
      • Added 6mL of water to the bacterial cell pellet.
  • Midiprepped DNA is stored at -20°C in the BioCurious freezer.