Testing mixing of pSPIN-BAC bacteria and DiNV DNA at Room Temp without Electroporation

I wondered wether the change from ice to warm buffer made the virus DNA go inside the bacteria cells (kinda like a heat shock) even without the electroporation and that was why all samples showed virus positive even if no electro treatment (previous tests). So if I did the process at room temp then maybe that wouldn’t happen.

Mixing

  • Prepared 2 tubes of 975ul SOC buffer to room temp
  • Using last of exo-20 sample and a 1:10 diluted exo-20 sample
  • Warmed LB and pSPIN-BAC cells to room temp
  • pSPIN-BAC DNA no electro - tube 12A and 12B
    • Added 25ul pSPIN-BAC cells to a tube 12A
    • Added 2ul 20-exo DNA
    • Immediately added 975ul of room temp SOC buffer and mix
    • Transferred 490ul to a 1.5mL tube labeled “12B”
    • Keep 12B rt
    • Placed 12A in the 30C shaking incubator 1 hour
  • pSPIN-BAC diluted DNA no electro - tube 13A and 13B
    • Added 25ul pSPIN-BAC cells to a tube 13A
    • Added 2ul 1:10 diluted 20-exo DNA
    • Immediately added 975ul of room temp SOC buffer and mix
    • Transferred 490ul to a 1.5mL tube labeled “13B”
    • Keep 13B rt
    • Placed 13A in the 30C shaking incubator 1 hour

Washing Cells

  • Centrifuged B tubes 3 min at 6,000g
  • Removed supernatant
  • Resuspended pellets in 200ul LB
  • Repeated above steps 4 more times for 5 washes
  • Removed final supernatant and froze pellets of bacteria at -80C
  • For A tubes after their 1 hour incubation, I also did 5 washes with 200ul of LB
  • These tubes were not frozen and immediately went to DNA extraction

DNA extraction

  • Used both the A and B tubes of bacteria cell pellets
  • B tubes were taken from -80 and thawed on ice
  • DNA extraction was done exactly as described in a previous post

PCRs on DNA Extracts

  • The point was test all of the DNA samples for if they have retained DiNV DNA with PCR
  • 4 PCRs were run on the samples, and the process followed the general PCR protocol completely. Master mix volumes are listed here:
reagent p47 16S lef 9 lef 4
GoTaq 32.5ul 32.5ul 32.5ul 32.5ul
F primer 1.625ul 1.625ul 1.625ul 1.625ul
R primer 1.625ul 1.625ul 1.625ul 1.625ul
molecular grade water 22.75ul 22.75ul 22.75ul 22.75ul
  • All PCR programs were run for 30 cycles, and program information can be found here
  • 1% gel was run at 90V for 45 minutes to resolve the bands: