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

The first attempt of this did not end up working, some samples with the colony PCR seemed at first to amplify p47, but when grown up in larger volumes and extracted normally did not. I am not sure if they lost it or never had it. Rob also reminded me that the Sternberg lab had more sucess with large insertions (the BAC is ~7,000pbp) when the bacteria were kept at 30C, so I will do that for every step this time.

I also decided to use less bacteria, 25ul of the electrocompetent cells compared to 50 because there were so many cells before that almost overloaded plates and this way I can make the electrocompetent bacteria last longer. This is also the volume recommended by NEB to use so it is not an unreasonable volume.

I used DNA from sample 22-exo (prepared here) and the electrocompetent pSPIN-BAC bacteria prepared here. I am also trying non-exonuclease treated DNA as well, sample 21 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 for the 22-exo sample. 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. The second positive sample 21 has higher DNA concentration around 100ng/ul, so I used 1ul of that DNA.

  • Prepared 4 1.5mL tubes with 9750ul of SOC medium
  • Thawed 2 tubes of electrocompetent cells on ice
  • Placed sample 22-exo and 21 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
    • 1.5mL tubes
    • Tubes with SOC
  • Placed the SOC tubes in their 30C incubator to warm up
  • Placed 3 electroporation cuvettes on ice to chill
  • Neg control 1:
    • Added 25ul of electrocompetent cells in a cold cuvette avoiding bubbles
    • Electroporated cuvette on EC2 settings
    • Immediately added ~975ul of warm SOC buffer and pipette mixed
    • Transferred the ~1000ul to a 1.5mL labeled “Neg 1” and placed in their 30C shaking incubator for 1 hour
  • Neg control 2:
    • Added 25ul of electrocompetent cells to a 1.5mL tube labeled “Neg 2”
    • Added 2.5ul of sample #22-exo DNA to the tube
    • Immediately added ~975ul of warm SOC buffer and pipette mixed
    • Placed in 30C shaking incubator for 1 hour
  • DiNV-1:
    • Added 25ul of electrocompetent cells to a 1.5mL tube on ice
    • Added 2.5ul of sample #22-exo DNA to the tube
    • Pipetted the tube once to mix
    • Transferred the sample mix to a chilled cuvette
    • Electroporate on EC2 settings
    • Immediately added ~975ul of warm SOC buffer and pipette mixed
    • Transferred the ~1000ul to a 1.5mL labeled “DiNV-1” and placed in their 30C shaking incubator for 1 hour
  • DiNV-2:
    • Added 25ul of electrocompetent cells to a 1.5mL tube on ice
    • Added 1ul of sample #21 DNA to the tube
    • Pipetted the tube once to mix
    • Transferred the sample mix to a chilled cuvette
    • Electroporate on EC2 settings
    • Immediately added ~975ul of warm SOC buffer and pipette mixed
    • Transferred the ~1000ul to a 1.5mL labeled “DiNV-2” and placed in their 30C shaking incubator for 1 hour
  • During the incubation, prepared the plates:
    • 16 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 175ul of stock chlor with 175ul of 100% ethanol
    • Added 20ul to each of 16 plates and spread them with a sterile spreader made from a pasture pipette
    • Placed plate in the 30C incubator to warm
  • Prepared 1 1.5mL tube with 1mL of LB and warmed in the 370C 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 150ul 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 100ul neg 1
4 10ul neg 2
5 40ul neg 2
6 100ul neg 2
7 30ul DiNV-1
8 30ul DiNV-1
9 30ul DiNV-1
10 30ul DiNV-1
11 30ul DiNV-1
12 30ul DiNV-2
13 30ul DiNV-2
14 30ul DiNV-2
15 30ul DiNV-2
16 30ul DiNV-2
  • Placed the plates upside down in the 30C incubator overnight to grow