Using the Hirt Extracted and Exonuclease Cleaned up DiNV DNA to Electroporate into pSPIN-BAC E. coli

This is the main experiment for cloning DiNV, hopefully once the DiNV DNA is in the cells that already have the pSPIN-BAC the bacteria will insert the BAC into the DiNV genome and then be able to replicate the genome and keep it because of the chloramphenicol resistance.

I used DNA from sample 20 (prepared here) and the electrocompetent pSPIN-BAC bacteria prepared here.

I plan to use 2.5ul of DNA because that would be around 70ng of DNA and not too large of a volume to add to the cell mixture for electroporation. The goal is to have less than 3ul added to the cells I think. I want to do a negative sample that gets no DNA but gets the electroporation. And I want a second negative that gets the DNA but does not get electroporated.

  • Prepared 3 1.5mL tubes with 950ul of SOC medium
  • Thawed 3 tubes of electrocompetent cells on ice
  • Placed sample 20 on ice after flick mixing and spinning down
  • Things brought up to Chandler lab:
    • Ice bucket with bacteria and DNA
    • Pipettes and tips
    • 95% ethanol
    • Electroporation cuvettes EC2
    • 1.5mL tubes
    • Tubes with SOC
  • Placed the SOC tubes in their 37C incubator to warm up
  • Placed 2 electroporation cuvettes on ice to chill
  • Neg control 1:
    • Added 50ul of electrocompetent cells in a cold cuvette avoiding bubbles
    • Electroporated cuvette on EC2 settings
    • Immediately added ~950ul of warm SOC buffer and pipette mixed
    • Transferred the ~100ul to a 1.5mL labeled “Neg 1” and placed in their 37C shaking incubator for 1 hour
  • Neg control 2:
    • Added 50ul of electrocompetent cells to a 1.5mL tube labeled “Neg 2”
    • Added 2.5ul of sample #20 DNA to the tube
    • Immediately added ~950ul of warm SOC buffer and pipette mixed
    • Placed in 37C shaking incubator for 1 hour
  • DiNV:
    • Added 50ul of electrocompetent cells to a 1.5mL tube on ice
    • Added 2.5ul of sample #20 DNA to the tube
    • Flicked to mix the tube and spun down
    • Transferred the sample mix to a chilled cuvette
    • Electroporate on EC2 settings
    • Immediately added ~950ul of warm SOC buffer and pipette mixed
    • Transferred the ~100ul to a 1.5mL labeled “DiNV” and placed in their 37C shaking incubator for 1 hour
  • During the incubation, prepared the plates:
    • 13 total plates going to be used
    • 50,000ug.mL * X = 20ug/mL * 25mL
    • X = 0.01mL or 10ul per plate of stock chloramphenicol needed
    • I want to have 20ul to add to spread on the plates
    • Diluted 140ul of stock chlor with 140ul of 100% ethanol
    • Added 20ul to each of 13 plates and spread them with a sterile spreader made from a pasture pipette
    • Placed plate in the 37C incubator to warm
  • Prepared 1 1.5mL tube with 1mL of LB and warmed in the 37C incubator
  • After 1 hour, took the tubes down to 4012
  • Centrifuged tubes for 3 min at 3,000rpm
  • Removed the supernatant to a 15mL conical and disposed of in the bacteria waste
  • Resuspended each pellet in 200ul of warm LB medium
  • Spread on the chloramphenicol resistance plates with a sterile loop spreader:
plate volume sample
1 10ul neg 1
2 40ul neg 1
3 150ul neg 1
4 10ul neg 2
5 40ul neg 2
6 150ul neg 2
7 30ul DiNV
8 30ul DiNV
9 30ul DiNV
10 30ul DiNV
11 30ul DiNV
12 20ul DiNV
13 30ul DiNV
  • Placed the plates upside down in the 37C incubator overnight to grow