Cloning human alpha casein S1, 04Oct2014 and on

Cloning human alpha casein S1, plasmid DNA midiprep

Overview

During these experiments, we will insert our final IDT gBlock, human alpha casein (received 03Oct2014,) into the pD1214 vector and transform our constructs into E. coli. We will then perform midiprep plasmid DNA extractions, quantify our plasmid DNA and submit plasmid DNA from duplicate clones for sequencing. Sequence-confirmed DNA is then ready to be transformed into yeast.

Cloning casein & FAM20C gBlocks into pD1214

October 4, 2014

• Participants: Meenakshi, Johan, Lafia

• Notes: Lafia, transcribed by Rachel

• Location: BioCurious

• Materials & Methods, Electra Cloning:
  • We resuspended lyophilized IDT gBlock DNA for P.FAKS.humAlphaS1.S, received 04Oct2014, as in DNA Handling using 0.5X TE (1X TE, pH 7.5: 1 diH2O). Final DNA concentration = 20ng/uL, per Electra cloning kit requirements.
  • Used DNA 2.0 Electra cloning kit protocol.

Reaction:

1uL pD1214 (20ng)
1uL of appropriate casein gBlock or (+) control DNA (20ng), or 0.5X TE (-) control
2uL Electra Buffer
1uL of Electra enzyme mix
15uL of diH20
----
20uL
Incubated 20 min room temperature

Transforming human alpha casein S1:pD1214 cloning reaction product into E. coli

October 4, 2014

• Participants: Lafia, Meenakshi, Johan

• Notes: Lafia, transcribed by Rachel

• Location: BioCurious

• Materials & Methods:
  • 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 3 reactions: one for human alpha casein S1 Electra cloning reaction (above,) one pGLO positive control, one competent cells-only negative control.
    • Thawed individual tubes of Mix & Go chemically competent, dH5alpha E. coli cells slowly on ice.
    • For each transformation reaction, prewarmed LB agar plates with Carb-50, 100mm (Teknova), at 37°C.
      • Inducible promoter for pGLO positive control requires arabinose, so used a plate that already contained [need concentration] of arabinose for that reaction.
    • Added 5uL of human alpha casein S1:pD1214 or appropriate control to individual thawed tube of 100uL competent cells. Mixed gently.
    • Immediately incubated on ice for 30 min.
    • In UV- & 70% isopropanol-sterilized laminar flow hood, plated 105uL of each reaction to LB carb plates, using one individually wrapped spreader/reaction.
    • Incubated upside down plates overnight at 37°C.

October 5, 2014

• Results, Discussion, Next Steps:
  • No colonies on any plate, perhaps due to lack of -80°C storage freezer for competent cells at BioCurious (cells always stored at -20°C). Will reorder Zymo Mix & Go competent dH5alpha E. coli cells and repeat.