Midiprep Plasmid Extraction of pAc5-EGFP and pBS-99F8-5’HR-attP»Act5C::dsRed<

I needed more pAc5 plasmid and we just purchased the pBS-99F8-5’HR-attP»Act5C::dsRed< (or 1544 dsRed plasmid as I’ll call it). Only the 1544dsRed plasmid is plated from an agar stab.

20230418 Plating Agar Stab

  • Warmed 2 LB plates on the bench, each with 100ug/mL Ampicillin (Kistie had made these previously)
  • Sterilized the bench space and the loop before use
  • Labeled 2 plates with the plasmid information
  • Dipped the loop in the 1544dsRed agar stab and spread on an LB plate
  • Sterilized the loop between each plate
  • Plates were placed upside down in the 37C incubator overnight, then put in the fridge the next day

20230419 Overnight Culture

  • In the afternoon, the best 1544dsRed plate with visible individual colonies was used to grow overnight cultures
  • Labeled 3 LB tubes
  • Sterilized the bench space and used the flame
  • Diluted Ampicillin:
    • Want 100ug/mL in 2mL
    • (100,000mg/mL)(x) = 100mL * 2mL
      • x = 0.002mL or 2ul
  • Added 2ul of Ampicillin stock to each LB tube
  • Picked 1 colony from the plate with a pipette tip and dropped it in the test tube
  • Placed the tubes in the 37C incubator shaking overnight

20230420 Making Glycerol Stock for 1544deRed

  • Sterilized the bench space and used the flame
  • Made up 2 cryotubes for making stocks
    • 143: pBS-99F8-5’HR-attP»Act5C::dsRed<
  • Added 750ul of 50% glycerol to each cryotube
  • Added 750ul of culture to the tubes and pipette mixed
  • Bleached the test tubes when done
  • Tubes went into the bacterial stocks and the back up stocks boxes in the -80

20230423 Making Overnight Cultures from Stock Grown Plates

  • Want to make 150mL of culture for both plasmids
  • Made 300mL of LB broth:
    • 6g LB
    • 3000mL DI H20
    • Shake to mix
  • Aliquoted 150mL into 2 500mL flasks
  • Foiled the flasks and autoclaved them on cycle 3
  • Put them in the fridge when done
  • Took them out before use to warm up a little
  • Calculated Ampicillin needed:
    • (100,000ug/mL)(x) = 100ug/mL * 150mL
      • x = 0.15mL or 150ul
  • Added 150ul of Ampicillin stock to the liquid LB and swirled it around
  • Picked 1 colony from the pAc5 plate and dropped it into the first flask
  • Picked 1 colony from the 1544dsRed plate and dropped it into the second flask
  • Placed the flasks in the 37 degree shaking incubator overnight

20230424 Midiprep Extractions

  • Started at 9am
  • Took flasks 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:
      • 4mL per conical, so 24mL buffer P1
      • 8ul 100mg/mL RNaseA, so 48ul
        • but, there was only 10ul left of that RNase A, so I also added 60ul of 10mg/mL RNase A (that’s not enough but hopefully it worked a little bit)
  • Got tubes from the Egan lab and poured off the supernatant
    • 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 50mL tubes
  • Centrifuged the 50mL tubes for 40 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 2 genomic tips over a 50mL conical for waste liquid
  • Added 4mL buffer QBT to the tips and let drip through
  • Brought tubes downstairs from Egan lab (turned off their centrifuge)
  • Transferred supernatant to the tips (one tip per plasmid) and let drip through, repeating for each tube, all liquid going into the same tip for each plasmid. I let the liquid completely drip out before adding in more
  • Transferred waste conicals when needed
  • Added 10mL of buffer QC to the tips and let drip through
  • Added another 10mL buffer QC and let drip through
  • Transferred the tips to a new 15mL tube
  • Added 5mL of warm buffer QF to the tips and let drip through
  • Prepared 7 1.5mL tubes and labeled as final tubes for each plasmid
  • 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: 315ul
      • dsRed: 370ul
  • 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: 220.5ul
      • dsRed: 259ul
  • Inverted tubes to mix
  • Centrifuged tubes for 45 minutes at 4C in the small centrifuge, 13,000rcf
    • Afterwards, I was able to see pellets!
  • 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 hrs on the bench
  • Resuspended pellets in each tube with 100ul 10mM Tris HCl
  • Let tubes incubate on the bench overnight

Qubit 20230425

  • Qubit results:
    • pAc5 1 = 51.4ng/ul
    • pAc5 2 = 47.1ng/ul
    • pAc5 3 = 50.2ng/ul
    • pAc5 4 = 47.6ng/ul
    • pAc5 5 = 43ng/ul
    • pAc5 6 = 44.7ng/ul
    • pAc5 7 = 12.1ng/ul
    • dsRed 1 = 316ng/ul
    • dsRed 2 = 11.6ng/ul
    • dsRed 3 = 291ng/ul
    • dsRed 4 = 260ng/ul
    • dsRed 5 = 268ng/ul
    • dsRed 6 = 304ng/ul
    • dsRed 7 = 112ng/ul