Plasmid Midiprep Extraction of pAc5 EGFP

The last plate I had made from this plasmid was many months ago, so I decided to make a new plate.

20230802 Plating from Glycerol Stocks

  • Labeled 2 LB plate
  • Added 20ul of stock Ampicillin to the plates, and spread it around with a sterile spreader (made these from glass pipettes)
  • Waited about 5 minutes for the antibiotic to soak in
  • Sterilized loop
  • Dipped the loop in the stock and spread it on the plate, and repeated for the second plate
  • Plates were put upside down in the 37C incubator and taken out and put into the fridge the next morning

20230807 Making Overnight Cultures from Stock Grown Plates

  • Want to make 150mL of culture for plasmid
  • Made 150mL of LB broth:
    • 3g LB
    • 150mL DI H20
    • Shake to mix
  • Aliquoted 150mL into 1 500mL flask
  • Foiled the flasks and autoclaved them on cycle 3
  • Put it on the counter when done
  • Added 150ul of Ampacillin stock to the liquid LB and swirled it around
  • Picked 1 colony from the 08/02 plate and dropped it into the flask
  • Placed the flask in the 37 degree shaking incubator overnight

20230808 Midiprep Extraction

  • Started at ~9am
  • Got access to the Egan lab with a key now, I just have to email Dr. Egan a few days before to let her know I want to use the centrifuge Took flask out of the incubator and used serological pipettes to transfer the liquid cultures into 50mL conicals
  • Took these tubes to the Egan lab and fast cooled their centrifuge. Then ran it at 6,000rcf at 4C for 20 minutes
  • While the centrifuge was running:
    • Got an ice bucket and chilled buffer P3
    • Prepared buffer P1: 12mL of P1 (mix before pipetting) and 120ul of 10mg/mL of RNase A and kept on ice
  • Got tubes from the Egan lab and poured off the supernatant into conicals that would then get put in the autoclave trash
    • There was a large bacterial pellet in all tubes
  • Added 4mL of buffer P1 and vortexed until the cell pellet was resuspended
  • Added 4mL of buffer P2 to each tube
    • Tubes should be gently mixed until all liquid becomes blue and viscous
  • Incubated tubes at room temp on the bench for 5 minutes
  • Added 4mL cold buffer P3 to each tube
    • Inverted tubes to mix until the liquid was white and not viscous
  • Incubated tubes on ice for 15 minutes
  • Took tubes to the Egan lab an centrifuged 4C at 12,400rcf for 40 minutes
  • Brought up new 15mL tubes to the Egan lab
  • By the centrifuge, transferred the supernatant to the new 15mL tubes
  • Centrifuged the 15mL tubes for 30 min at 4C 12,400rcf
  • Put the small centrifuge in the fridge to cool down
  • Turned on the incubator to 65C and warmed buffer QF
  • Prepared a new conical of 100% isopropanol and 70% ethanol
  • Set up a genomic tips over a 50mL conical for waste liquid
  • Added 4mL buffer QBT to the tip and let drip through
  • Brought tubes downstairs from Egan lab (turned off their centrifuge)
  • Transferred supernatant to the tip and let drip through, repeating for each tube, all liquid going into the same tip. I let the liquid completely drip out before adding in more
  • Transferred waste conical when needed
  • Added 10mL of buffer QC to the tip and let drip through
  • Added another 10mL buffer QC and let drip through
  • Transferred the tip to a new 15mL tube
  • Added 5mL of warm buffer QF to the tip and let drip through
  • Prepared 7 1.5mL tubes and labeled as final tubes
  • Took the eluted liquid from the 15mL conical and spread it out over the 7 1.5mL tubes
    • For the first 6 tubes they would get 833ul elutent each
    • The 7th tube got a different amount of liquid (whatever was left):
      • pAc5: 335ul
  • Added 0.7 volumes 100% isopropanol to each of the 1.5mL tubes
    • For 833ul volume tubes, that is 583ul isopropanol
    • For the 7th tube that is:
      • pAc5: 235ul
  • Inverted tubes to mix
  • Centrifuged tubes for 45 minutes at 4C in the small centrifuge, 13,000rcf
    • Afterwards, I was able to see pellets in most tubes!
  • Decanted off supernatant in the 1.5mL tubes
  • Added 500ul of cold 70% ethanol to each tube
  • Centrifuged tubes 30 minutes at 4C 13,000rcf
  • Poured off ethanol and let tubes dry ~2 hours minutes on the bench
  • Resuspended pellets in each tube with 50ul 10mM Tris HCl
  • Let tubes incubate on the bench overnight

Qubit 20221028

  • Qubit results:
    • pAc5 1 : 810ng/ul
    • pAc5 2 : 848ng/ul
    • pAc5 3 : 8.74ng/ul
    • pAc5 4 : 764ng/ul
    • pAc5 5 : 744ng/ul
    • pAc5 6 : 698ng/ul
    • pAc5 7 : 260ng/ul

It looks like I lost a pellet for number 3, but the others look great! This is a lot of plasmid to work with.