Plasmid Mini-prep and Preparation for Sequencing of pSPIN-BAC

We want to sequence the pSPIN-BAC just to make sure that it is the same plasmid that Genewiz sent us and their sequence they sent was correct.

Based off of the previous work that showed that colony 3 bacteria is not resistant to Chloramphenicol, I decided to use a colony from the chlor+kan plate from colony 1 to grow up for sequencing.

20231217 growing overnight cultures

  • Prepared 3 tubes of LB with both antibiotics:
    • 0.9ul of stock Kanamycin
    • 0.55ul of stock Chloramphenicol
    • These are based off of the tubes of LB being slightly less than 2mL and previous volumes used
  • From the chlor+kab plate of the colony 1 stock, a colony was picked and placed into each tube with a tip
  • The tubes were placed in the 37C shaker overnight to grow

20231218 Plasmid Prep

  • Using the Qiagen Qiaprep Miniprep for quick plasmid extraction, these will just be for sequencing and don’t need a lot or endotoxin free
  • Placed an aliquot of elution buffer in the heat block at 70C
  • All 3 culture tubes had grown nicely
  • Harvested 4mL of culture, 1mL into each of 4 1.5mL tubes
  • Centrifuged the tubes for 3 minutes at 6,000g
  • Removed the supernatant from each tube into the bacteria waste
  • Resuspended the pellets in 250ul of cold buffer PI (stored in 4C) until there were no more cell clumps
  • Added 250ul of buffer P2 and inverted to mix, solution became sort of viscous
  • Added 350ul of buffer N3 and mixed immediately 1 tube at a time by inverting
    • Visible precipitate was seen
  • Centrifuged at 16,300rcf for 10 minutes at room temp
    • this is to pellet the debris, SDS, and chromosomal DNA
  • Added the ~800ul of supernatant to the spin columns provided by the kit
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Added 500ul of buffer PB to the columns
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Added 750ul of buffer PE to the columns
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Discarded the flow through
  • Centrifuged the columns 60 seconds at 16,300rcf “dry”
  • Placed spin columns in final 1.5mL tubes labeled
  • Added 30ul of warmed buffer EB and let sit on the filter for 1 minute
  • Centrifuged the columns 60 seconds at 16,300rcf
  • Placed tubes with DNA on ice and quantification
  • Quantified the DNA with the Qubit using the standard Qubit protocol
    • 1: 36.3ng/ul
    • 2: 47.1ng/ul
    • 3: 43ng/ul
    • 4: 27.9ng/ul
  • For Plasmidsaurus they want at least 10ul with a concentration of 30ng/ul
  • I decided to use tube 2 because it had the most DNA
  • I wanted to send 20ul, at 30ng/ul that is a total of 600ng to send
  • 600/47.5 = 12.6ul of tube 2 and 7.4ul of buffer EB to dilute the DNA
  • This was put in a strip tube, wrapped in parafilm, and placed in a 50mL conical
  • The strip tube had the code for sequencing A4W on it
  • Because of the holidays, the sample was not sent out for sequencing until Jan 2nd, and was in the -20 until then
  • Sequencing showed that the consensus sequence from Plasmidsaurus was an exact match to the sequence sent from Genewiz, plasmid is intact and ready to use