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.