Using Qiagen Plasmid Midi Prep Kit to Extract pBeloBAC11 and pBACe3.6, Using 25mL Overnight Culture

Notes

  • Used 25mL of LB, used the same streaked plate as last week to pick a colony
  • Used new centrifuge tubes that had a max speed of 12,500g, I used 12,400g for spins to not strain them

20220214 Making LB and Overnight Culture

  • Prepared 250mL of LB
    • 5g LB broth base in 250mL of distilled water, shaken until homogenous
  • Aliquoted 25mL of LB into 2 new 250mL flasks that had been previously autoclaved
  • Used the rest of the LB to make a new rack of 2mL LB test tubes
  • Covered the flasks with foil
  • Autoclaved in program 3
  • Put flasks in the fridge after
  • Later that day, placed the flasks on the bench for a while to warm
  • Labeled each flask with tape, either pBeloBAC11 or pBACe3.6
  • Started the bunsen burner
  • Added 7.5ul of 50mg/mL of Chloramphenicol to each flask
  • Picked 1 colony from each streak plate with a p2 pipette tip, and dropped the tip into the appropriate flask
  • Covered the flask with the same foil and placed in the 37 degree C shaking incubator overnight

20220215 MidiPrep Extraction

  • Transferred volume from flasks (~25mL) to green new 50mL conicals
  • Took upstairs to the Egan Lab and centrifuged 6,000rcf, 4 degrees C, 20 minutes
  • Got ice and put buffer P3 on ice to chill
  • Prepared buffer P1
    • Made an aliquot of 8mL of P1
    • Added 16ul of 100mg/ul RNase A to the aliquot (for a final concentration of 100ug/mL)
    • Kept the tube on ice
  • Removed supernatant from the bacterial pellets after centrifugation
  • Added 4mL of buffer P1 to each tube and vortexed until there was no longer a pellet or any clumps
  • Added 4mL of buffer P2 to each tube
    • Liquid became blue and the tubes were inverted to mix until all the liquid was blue and there were no longer any clumps - very viscous
  • Tubes were incubated at room temp for 5 minutes
  • Added 4mL of chilled buffer P3 to each tube
    • Inverted to mix tubes until there was no longer any blue and any viscosity
    • Lots of white precipitate showed up in the liquid
  • Placed the tubes in the ice for 15 minutes
  • Took the tubes upstairs to the Egan lab and centrifuged 40 min at 12,400rcf and 4 degrees C
  • After: brought new 15mL tubes upstairs and a p1000 pipette and tips
  • Beside the centrifuge, pipetted the supernatant off into new 15mL tubes for each tube (there was a huge white pellet)
  • Centrifuged the supernatant tubes for 30 minutes at 12,400rcf and 4 degrees C
  • While that was going, made a new conical of 100% isopropanol and 70% ethanol
  • Set up two genomic tips over 50mL conicals and labeled with tape
  • Added 4mL of buffer QBT to each tip and let it drip through to equilibrate
  • Warmed the incubator to 65 degrees C and put buffer QF inside to warm
  • Removed tubes from the centrifuge and transferred the supernatant to new 15mL tubes (there was hardly any pellet this time)
  • Transferred the supernatant liquid to their respective tips and let the liquid drip through
  • Transferred to a new 50mL conical for waste
  • Added 10mL of buffer QC to each tip and let drip through
  • Added another 10mL of buffer QC to each tip and let drip through
  • Transferred the tips to new 15mL tubes labeled as final tubes
  • Added 5mL of warmed buffer QF to each tip and let drip
  • Added 3.85mL of 100% isopropanol to each tube and inverted to mix (tubes had ~5.5mL volume, 0.7 volumes is 3.85mL)
  • Centrifgued tubes for 45 minutes at 12,400rcf at 4 degrees C. Made sure they were facing a certain way to look for the pellet
  • Looked for pellets afterwards - pretty sure that these clear streaks are the pellet, they went away after hydration this time
  • Decanted the supernatant next to the centrifuge in the Egan lab
  • Added 2mL of 70% ethanol to each tube
  • Placed tubes back in the centrifuge for 30 min, 12,400rcf at 4 degrees C, same tube orientation
  • Poured off the ethanol and let the tubes air dry for ~10 minutes
  • Added 200ul of Qiagen elution buffer (10mM Tris HCl pH 8.5) to where I thought the pellet might be, and tried to keep the liquid bubble on those streaks
  • Placed the tubes on their sides in the 65 degree incubator for ~1hr
  • Qubit:
    • pBeloBAC11: 4.77ng/ul
    • pBACe3.6: 41.7ng/ul

In theory, I maxed out the kit with the 25mL culture volume, but clearly I did not get the maximum yeild of DNA: 100ug. Total DNA is 954ng for pBeloBAC11 and 8.340ug for pBACe3.6. This is enough DNA for pBACe3.6, I need 5ug to send to Genewiz. But I am still very low for pBeloBAC11.
If we compare these yields to my previous attempt, I can calculate “expected yield”.
For pBeloBAC11 I got 111ng total yield from 4mL culture. 25mL/4mL is 6.25. 111 x 6.25 = 693.75ng expected yield. So I got more than that this time.
For pBACe3.6 I got 2.38ug total. 2.38 x 6.25 is 14.875ug expected yield. I got a little over half that for this extraction.