Growth, DNA and Plasmid Extraction, and PCR from 5 Colonies from 1st Electroporation Attempt

Colonies 38, 40, 42, 46, and 49 from the colony PCR of the first electroporation attempt looked promising like they might be p47 promising. 2mL LB cultures were made from each colony the afternoon before. The 2mL cultures were first mixed with 0.8ul of stock chloramphenicol. Then the remaining ~2ul from the liquid that went into the colony PCR was used to seed each culture tube. Each sample got 1 tube. These were grown shaking overnight at 37C.

DNA Extraction

  • A dip of a sterile loop of each colony was plated on a plate with chloramphenicol to have a stock of the bacteria tested and grown overnight at 37C just in case
  • Heat block warmed to 37C
  • Cell lysis solution chilled on ice until cloudy
  • 500ul of overnight solution per tube was added to a 1.5mL tube
  • Tubes were spun down for 30 seconds at 13,000rpm
  • Supernatant was discarded
  • Added 300ul of chilled cell lysis solution
  • Added 0.6ul of 10mg/mL RNase A and inverted 25 times and spun down
  • Place tubes on heat block at 37C for 1 hour
  • Prepared fresh 70% ethanol and final 1.5mL tubes with 350ul of 100% isopropanol
  • After incubation, placed tubes on ice
  • Added 150ul of protein precipitation solution
  • Vortexed tubes and placed on ice for 5 minutes
  • Centrifuged tubes for 5 min at max speed
  • Transferred supernatant (~450ul) to the final tubes with isopropanol and inverted 50X
  • Centrifuged tubes for 3 min at max speed
  • Discarded supernatant
  • Added 300ul fresh 70% ethanol and inverted twice
  • Centrifuged tubes 1 minute max speed
  • Discarded supernatant
  • Let tubes air dry on kim wipe ~30 min
  • Resuspended pellet in 100ul of DNA hydration solution - DNA amount was large and visible and let resuspend on bench for a while

Plasmid Extraction

  • Using the Qiagen Qiaprep Miniprep for quick plasmid extraction
  • Placed an aliquot of elution buffer in the heat block at 70C
  • Harvested 1mL of culture from each tube into a 1.5mL tube (1 tube per colony)
  • Centrifuged the tubes for 3 minutes at 6,000g
  • Removed the supernatant from each tube into the bacteria waste
  • Resuspended the pellets in 250ul of cold buffer PI (stored in 4C) until there were no more cell clumps
  • Added 250ul of buffer P2 and inverted to mix, solution became sort of viscous
  • Added 350ul of buffer N3 and mixed immediately 1 tube at a time by inverting
    • Visible precipitate was seen
  • Centrifuged at 16,300rcf for 10 minutes at room temp
    • this is to pellet the debris, SDS, and chromosomal DNA
  • Added the ~800ul of supernatant to the spin columns provided by the kit
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Added 500ul of buffer PB to the columns
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Added 750ul of buffer PE to the columns
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Centrifuged the columns 60 seconds at 16,300rcf “dry”
  • Placed spin columns in final 1.5mL tubes labeled
  • Added 20ul of warmed buffer EB and let sit on the filter for 1 minute
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Kept DNA on ice

p47 and 115 PCRs

  • Thawed reagents and primer on ice, vortexed and spun down
  • Want to do each samples with both primers, and use positive and negative controls
  • Made master mix on ice:
reagent p47 volume 115 volume
GoTaq 75ul 75ul
F primer 3.75ul 3.75ul
R primer 3.75ul 3.75ul
molecular grade water 52.5ul 52.5ul
  • Vortex and spun down master mix
  • Added 9ul of mix to their appropriate strip tubes
  • Added 1ul of DNA to their appropriate tube
  • Added 1ul of molecular grade water for the negative
  • Used a positive sample for the positive
  • Vortexed and spun down tubes
  • Placed tubes in the 115 and p47 PCR programs (35 cycles each)

Afterwards a 1% gel was run for 35 minutes at 90V:

It did not look to me that any sample had the appropriate amplification to indicate virus presence (I suspect some off target amplification in the 115 set), so these samples were not considered again. Sample/colony information can be found here