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 P.FAKS.humAlphaS1.S gBlocks into pD1214vector

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

• 'Aims: To get human alpha casein S1:pD1214 into the dH5alpha E. coli strain.

• 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.
    • Protocol change: Due to pipetting error, accidentally added all 20uL of Electra cloning reaction, above, to thawed tube of 100uL competent cells, a 1:5 ratio of cloning rxn:cells, rather than the usual 1:20. Added another 100uL competent cells, bringing ratio to 1:10. Mixed gently. Positive control = 5uL pGLO plasmid DNA in one 100uL thawed tube competent cells.
    • 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 for controls. Divided transformation 220uL for plated half onto each of two plates.
    • Incubated upside down plates overnight at 37°C.

October 5, 2014

• Results, Discussion, Next Steps:
  • No colonies on any plate.
  • Trouble-shooting: perhaps due to ratio change, above, reduction in Electra enzyme functionality after many times in and out of the -20°C freezer, or 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.

Re-cloning P.FAKS.humAlphaS1.S gBlocks into pD1214vector

October 4, 2014

• Participants: Meenakshi, Johan

• Notes: Johan, transcribed by Rachel

• Location: BioCurious

• Aims: As October 4, 2014 transformation of P.FAKS.humAlphaS1.S:pD1214 to dH5alpha, above, failed, repeating Electra cloning of P.FAKS.humAlphaS1.S to pD1214, as all Oct. 4 reaction volume mistakenly used in previous transformation. Reaction products will be used to repeat transformation with new Zymo Mix & Go chemically competent dH5alpha E. coli.

• Materials & Methods:
  • We used the P.FAKS.humAlphaS1.S gBlock DNA, resuspended Oct. 4, above.
  • 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

Re-transforming human alpha casein S1:pD1214 cloning reaction product into E. coli

October 7, 2014

• Participants: Meenakshi, Johan

• Notes: Johan, transcribed by Rachel

• Location: BioCurious

• Aims: Repeat above Oct. 4 failed transformation of P.FAKS.humAlphaS1.S:pD1214 into dH5alpha E. coli with new Zymo Mix & Go chemically competent cells, in case transformation failure due to previous cells' extended storage at -20°C, rather than -80°C.

• Materials & Methods:
  • Used new Zymo Research "Premade Mix & Go Competent E. coli Cells" received since October 4.
  • 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 Oct. 7 Electra cloning reaction product, above, or pGLO positive control plasmid to in one 100uL thawed tube of competent cells. Negative control = one cell tube only, otherwise, treated identically.
    • Immediately incubated all tubes 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 for controls.
    • Incubated upside down plates overnight at 37°C.

October 8, 2014

• Results:
  • P.FAKS.humAlphaS1.S:pD1214 = 3 colonies
  • pGLO (+) control = 1 colony
  • cells only (-) control = 0 colonies

• Discussion:
  • Colony counts extremely low!
  • Failed Oct. 4 transformation not likely due to, or solely due to, extended length of time competent cells were stored at -20°C or altered transformation reaction ratios.
  • Poor Electra enzyme mix functionality likely, as enzyme tube has been in and out of freezer for many reactions, and has been left on ice on benchtop for longer than required by Electra protocol.

• Next Steps:
  • Grow up colonies for midiprep of P.FAKS.humAlphaS1.S:pD1214.
  • Must improve enzyme handling procedures: reduce extended time enzymes have been spending on bench, even if on ice. Aliquot enzymes upon receipt? Obtain cooler rack for enzyme tubes for benchtop use?

Plasmid DNA midiprep of P.FAKS.humAlphaS1.S:pD1214

October 8, 2014

• Participants: Johan

• Notes: Johan, transcribed by Rachel

• Location: BioCurious

• Aims: Midiprep plasmid DNA from October 7 transformation, above, of P.FAKS.humAlphaS1.S:pD1214 to dH5alpha so can submit for sequencing.

• Materials and Methods:
  • Grew liquid cultures of P.FAKS.humAlphaS1.S:pD1214 to dH5alpha in LB carbenicillin, shaking, at 37°C.
  • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎) with the following specifics:
    • Followed "Centrifugation Protocol" stream .
      • 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 for sequencing. Remaining 140uL DNA stored in Johan's pink box, BioCurious -20°C freezer.

Notes on this construct continue: Submitting 4 repeat and 1 new casein in pD1214 plasmid for sequencing 10Oct2014