Second Attempt At Creating Glycerol Stocks from pSL1142 from Addgene, and Doing a Plasmid Midiprep to Extract Plasmid DNA

I received some information from Addgene on what Kanamycin concentration to use to keep the plasmid in the bacteria. They suggested using 50ug/mL for all culturing.

20221018 Plating Agar Stabs

  • Warmed 2 LB plates on the bench
  • Sterilized the bench space and the loop before use
  • Labeled 2 plates with the plasmid information
  • Diluted the 100mg/mL Kanamycin stock:
    • Want 50ug/mL in 20mL (plate volume)
    • (100,000mg/mL)(x) = 50mL * 20mL
      • x = 0.01mL or 10ul
    • Dilute 20ul Kanamycin in 40ul of water
  • Pipetted 30ul of diluted Kanamycin on each plate and spread with a sterile spreader
  • Waited ~3 minutes for it to soak in
  • Dipped the loop in the 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

20221020 Overnight Cultures

  • In the afternoon, the best plate with visible individual colonies was used to grow overnight cultures
  • Labeled 3 LB tubes
  • Sterilized the bench space and used the flame
  • Diluted Kanamycin:
    • Want 50ug/mL in 2mL
    • (100,000mg/mL)(x) = 50mL * 2mL
      • x = 0.001mL or 1ul
  • Added 1ul of Kanamycin 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

20221021 Making Glycerol Stocks

  • Sterilized the bench space and used the flame
  • Made up 2 cryotubes for making stocks
  • Going to replace the old glycerol stocks I had made that don’t actually have the plasmid
    • 137: pSL1142 pSPIN
  • 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

20221025 Plating from Glycerol Stocks

  • Labeled 1 LB plate
  • Added a diluted Kanamycin to the plate (10ul stock + 20ul water, as above) and spread with a sterile spreader
  • Sterilized loop
  • Dipped the loop in the stock and spread it on the plate
  • Plate was put upside down in the 37C incubator and taken out and put into the fridge the next morning

20221026 Making Overnight Cultures from Stock Grown Plates

  • Want to make 150mL of culture for pSL1142
  • Made 250mL of LB broth:
    • 5g LB
    • 250mL DI H20
    • Shake to mix
  • Aliquoted 150mL into 1 500mL flask
  • Used the rest for test tubes
  • 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 Kanamycin needed:
    • (100,000ug/mL)(x) = 50ug/mL * 150mL
      • x = 0.075mL or 75ul
  • Added 75ul of Kanamycin stock to the liquid LB and swirled it around
  • Picked 1 colony from the 10/25 plate and dropped it into the flask
  • Placed the flask in the 37 degree shaking incubator overnight

20220908 Midiprep Extraction

  • Started at ~12:20pm
  • 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 24ul 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 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):
      • pSL1142: 390ul
  • 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:
      • pSL1142: 273ul
  • 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 ~20 minutes on the bench
  • Resuspended pellets in each tube with 40ul 10mM Tris HCl
  • Let tubes incubate on the bench overnight

Qubit 20221028

  • Qubit results:
    • pSL1142 1 = 246ng/ul
    • pSL1142 2 = 4.64ng/ul
    • pSL1142 3 = 348ng/ul
    • pSL1142 4 = 370ng/ul
    • pSl1142 5 = 373ng/ul
    • pSL1142 6 = 367ng/ul
    • pSL1142 7 = 150ng/ul

It looks like I may have lost a pellet for tube 2, but the other concentrations are very high. This is a good sign that I actually got the plasmid this time. I want to run it on a gel to make sure. The plasmid size should be ~14,000bp.

20221108 gel

  • Because of the high concentration and the large size, it took a couple tries to get the gel conditions correct for visualizing the bands
  • I did various dilutions, and also used the previous (bad) extract to compare
  • This was a .9% gel run at 90V for 80 minutes