Creating Glycerol Stocks from pSL1142, pCS2+8CeGFP, and pAc5.1B EGFP from Addgene, and Doing a Plasmid Midiprep to Extract Plasmid DNA

Note: Even though these plasmids do have antibiotic resistance genes, I don’t know the concentration to use for them, so I did not use any antibiotics for any of this.

20220905 Plating Agar Stabs

  • Addgene sent us agar stabs for each plasmid, they were kept at 4C for a few days
  • Warmed 6 LB plates on the bench
  • Sterilized the bench space and the loop before use
  • Labeled 2 plates per plasmid
  • Dipped the loop in the agar stab and spread on an LB plate
  • Sterilized the loop between each plate
  • Made two plates per stab just in case one didn’t grow
  • Plates were placed upside down in the 37C incubator overnight

20220906 Overnight Cultures

  • In the morning, the plates were placed in the 4C fridge so they wouldn’t overgrow, each plate had good growth on them. They were wrapped in parafilm before going in the fridge
  • In the afternoon, the plate with visible individual colonies was used to grow overnight cultures
  • Labeled 3 LB tubes per plasmids
  • Sterilized the bench space and used the flame
  • Picked 1 colony from the plate with a pipette tip and dropped it in the test tube for each plasmid
  • Placed the tubes in the 37C incubator shaking overnight

20220907 Making Glycerol Stocks

  • Sterilized the bench space and used the flame
  • Made up 2 cryotubes per plasmid for making stocks
  • Added the plasmids to the bacterial stocks sheet
    • 137: pSL1142 pSPIN
    • 138: pCS2+8CeGFP
    • 139: pAc5.1B EGFP
  • Added 750ul of 50% glycerol to each cryotube
  • Added 750ul of culture to their respective 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

20220907 Plating from Glycerol Stocks

  • Immediately after the glycerol stocks were made, I plated from one of them
  • Labeled 2 LB plates per plasmid
  • Sterilized loop between each plate
  • Dipped the loop in the stock and spread it on the plate
  • Plates were put upside down in the 37C incubator
  • This was done at ~8am

20220907 Making Overnight Cultures from Stock Grown Plates

  • Need to know how much culture to grow for the midiprep extractions the next day:
    • pSL1142 is a low copy plasmid
    • pCS2 is a high copy plasmid
    • pAc5.1B is a high copy plasmid
  • I decided to do 150mL of culture for pSL1142, and 50mL each for the others
  • Made 250mL of LB broth:
    • 5g LB
    • 250mL DI H20
    • Shake to mix
  • Aliquoted 50mL into 2 125mL flasks
  • Aliquoted 150mL into 1 500mL flask
  • Foiled the flasks and autoclaved them on cycle 3
  • Put them in the fridge when done
  • Came back to the lab ~8pm to make the cultures (plates had grown enough by then to see colonies)
  • Picked 1 colony from the plate with a pipette tip and dropped it in the flask for each plasmid
  • Placed the flasks in the 37C incubator shaking overnight (foil lids were left loose)

20220908 Midiprep Extraction

  • Started at ~12:20pm
  • Took flasks out of the incubator and used serological pipettes to transfer the liquid cultures into 50mL conicals
  • Made 1 extra conical with ~50mL of water for a centrifuge balance
  • Took these tubes to the Egan lab and fast cooled their centrifuge. Then ran it at 6,000rcf at 4C for 20 minutes. Note: their centrifuge only has holders for 6 50mL conicals
  • While the centrifuge was running:
    • Got an ice bucket and chilled buffer P3
    • Prepared buffer P1: 20mL of P1 (mix before pipetting) and 40ul of 100mg/mL of RNase A and kept on ice
  • 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 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 3 genomic tips over 50mL conicals for waste liquid
  • Added 4mL buffer QBT to each and let drip through
  • Brought tubes downstairs from Egan lab (turned off their centrifuge)
  • Transferred supernatant to the appropriate tip and let drip through
    • There were 3 15mL tubes for pSL1142, so I added all the supernatants to the same tip. I would let one drip all the way through then add the next one
  • Transferred waste conicals when needed
  • Added 10mL of buffer QC to each tip and let drip through
  • Added another 10mL buffer QC and let drip through
  • Transferred the tips to new 15mL tubes
  • Added 5mL of warm buffer QF to each tip and let drip through
  • Prepared 6 1.5mL tubes per plasmid and labeled as final tubes
  • Took the eluted liquid from the 15mL conicals and spread it out over the 6 1.5mL tubes
    • For the first 5 tubes they would get 833ul elutent each
    • The 6th tube would get a different amount of liquid (whatever was left):
      • pSL1142: 560ul
      • pCS2: 600ul
      • pAc5: 490ul
  • Added 0.7 volumes 100% isopropanol to each of the 1.5mL tubes
    • For 833ul volume tubes, that is 583ul isopropanol
    • For the 6th tubes that is:
      • pSL1142: 392ul
      • pCS2: 420ul
      • pAc5: 343ul
  • 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 the pCS2 and pAc5 tubes, but not in the pSL1142 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 ~20 minutes on the bench
  • Resuspended pellets in each tube with 25ul 10mM Tris HCl
  • Let tubes incubate in the 65 degree incubator ~20 minutes then put in the fridge for overnight

Qubit 20220909

  • Note here, most the pCS2 and pAc5 samples read as too high on the Qubit, so I increased the volume in those samples by 100ul of 10mM Tris HCl and re-Qubited them
  • Reminder that there are 6 tubes per plasmid because of the way I did the precipitation
  • Qubit results:
    • pSL1142 1 = 20.8ng/ul
    • pSL1142 2 = 21.1ng/ul
    • pSL1142 3 = 18.4ng/ul
    • pSL1142 4 = 16.5ng/ul
    • pSl1142 5 = 23ng/ul
    • pSL1142 6 = 14.9ng/ul
    • pCS2 1 = 370ng/ul
    • pCS2 2 = 382ng/ul
    • pCS2 3 = 201ng/ul
    • pCS2 4 = 150ng/ul
    • pCS2 5 = 374ng/ul
    • pCS2 6 = 289ng/ul
    • pAc5 1 = 167ng/ul
    • pAc5 2 = 171ng/ul
    • pAc5 3 = 158ng/ul
    • pAc5 4 = 2.72ng/ul
    • pAc5 5 = 11.6ng/ul
    • pAc5 6 = 92.7ng/ul