Preparing pSL1142, pCS2+8EGFP, and pAc5.1B EGFP for Plasmidsaurus Sequencing

Plasmidsaurus has a drop box in Haworth at the Biostore that is picked up at 1pm on Mondays. For sequencing, they require at least 10ul of purified plasmid that is at 30ng/ul concentration.

Bead Clean pSL1142

  • None of my extracts for pSL1142 were at 30ng/ul or higher, so I decided to combine them all and bead clean and concentrate them
  • Took the beads out ~30 minutes before use, swirled to mix and left in a drawer to warm
  • Combined all extract tubes into 1 single tube, ~144ul volume total
  • Added 1X (144ul) beads to the sample tube and pipette mixed at least 10 times
  • Placed the tube on the orbital shaker 150rpm for 15 minutes
  • Prepared fresh 80% ethanol
  • Placed the tube on the magnet stand and waited for the liquid to go clear
  • Pipetted off most of the liquid without disturbing the beads
  • Added 500ul 80% ethanol to the tube
  • Waited ~30 seconds
  • Removed the ethanol
  • Added another 500ul 80% ethanol to the tube
  • Waited ~30 seconds
  • Removed all ethanol, using a small pipette for any droplets left
  • Waited ~5 minutes for the tubes to dry, but did not let the beads get dry
  • Resuspended the beads in 41ul 10mM Tris HCl
  • Placed the tube back on the shaker for 5 minutes
  • Placed the tube back on the magnet and waited until the liquid went clear
  • Removed 40ul into a new final tube
  • Qubit:
    • 52.6ng/ul

Prep Plasmid DNA for Sequencing

  • DNA was prepped the Friday before so it would be ready on the Monday
  • For pCS2 + 8EGFP and pAc5.1B EGFP I made 20ul of 30ng/ul plasmid
  • For pSL1142 pSPIN I made 12ul of 30ng/ul
  • pCS2 + 8EGFP:
    • 20ul of 30ng/ul is 600ng total DNA needed
    • 600ng/150ng/ul = 4ul plasmid DNA
    • 16ul 10mM Tris HCl
  • pAc5.1B GFP:
    • 20ul of 30ng/ul is 600ng total DNA needed
    • 600ng/92.7ng/ul = 6.47ul plasmid DNA
    • 13.53ul 10mM Tris HCl
  • pSL1142 pSPIN
    • 12ul of 30ng/ul is 360ng total DNA needed
    • 360ng/52.6ng/ul = 6.84ul plasmid DNA
    • 5.16ul 10mM Tris HCl

Sending for Sequencing 20220912

  • Liquid from these samples was pipetted into a 8 strip tube unlabeled along with some samples from Emma:
    • Tube 1 : pSL1142
    • Tube 2 : pCS2 + 8EGFP
    • Tube 3 : pAc5.1B GFP
    • Tube 4 : MW #35
    • Tube 5 : 35
    • Tube 6 : 41.2
    • Tube 7 : 41.3
  • Then the tubes were labeled with the 3 character code provided by the company
  • The caps were parafilmed, and the strip was put in a 50mL tube for transport
  • The invoice was printed and put in a plastic bag with the samples, which was brought to the dropbox in the Biostore