Synthesis of sgRNAs 86 and 92 for the 77kb DiNV Region Using the EnGen® sgRNA Synthesis Kit, S. pyogenes and Purification with the Zymo RNA Clean & Concentrator

Prep

  • p2 and p20-200 tips were autoclaved, and filter p1000 tips were acquired
  • The whole bench was wiped down with 70% ethanol and then RNase Away, as well as the tip boxes, pipettes, racks, and gloves
  • Thawed kit components on ice, as well as the guide RNA primers (designed here) that have been diluted to 1uM
  • Set the heat block to 37 degrees C

sgRNA Synthesis Kit

  • Flicked to mix tubes and spun them all down
  • Assembled separate reactions in 1.5mL tubes on the bench, not on ice
  • Each reaction contained (in order):
    • 2ul molecular grade water
    • 10ul 2X sgRNA reaction mix
    • 1ul of 1uM primer, either for 86 or 92
    • 1ul 0.1M DTT
    • 2ul sgRNA enzyme mix
  • Tubes were pipette mixed with 10ul
  • Tubes were spun down
  • Placed tubes in the heat block at 37 for 30 minutes
  • Afterwards, transferred the tubes to ice
  • Added 30ul molecular grade water to each tube
  • Added 2ul of DNaseI and pipette mixed
  • Spun down tubes
  • Incubated tubes in the heat block at 37 for 15 minutes

RNA Clean and Concentrator Kit

  • Took tubes out of the heat block and let come to room temp
  • Made sure RNA wash buffer had ethanol added (it had)
  • Added 2X the volume of sample (104ul) of RNA binding buffer to each tube
  • Pipette mixed
  • Added 1.5X the volume (231ul) 100% ethanol to each tube (the kit says to do 1.5X for RNA expected to be between 17-200nt in size, the NEB kit says they should be ~100nt)
  • Pipette mixed
  • Transferred liquid from each tube into their own spin column
  • Centrifuged columns at 15,800rcf for 30 seconds
  • Discarded flow through
  • Added 400ul RNA prep buffer to each column
  • Centrifuged columns at 15,800rcf for 30 seconds
  • Discarded flow through
  • Added 700ul RNA wash buffer to each column
  • Centrifuged columns at 15,800rcf for 30 seconds
  • Discarded flow through
  • Added 400ul RNA wash buffer to each column
  • Centrifuged columns at 15,800rcf for 1 minute
  • Discarded flow through
  • Centrifuged columns “dry” at 15,800rcf for 30 seconds
  • Transferred columns to final 1.5mL tubes
  • Added 40ul nuclease free water to each column and let sit for ~1 minute
  • Centrifuged columns at 15,800rcf for 30 seconds
  • Placed tubes on ice

Quantification

  • Used Qubit HS RNA kit
  • Made mix:
    • 895ul buffer
    • 4.5ul reagent
  • 190ul mix to standard tubes
  • 199ul mix to sample tubes
  • 10ul of standards to standard tubes
  • 1ul RNA to sample tubes
  • Vortex, spin down, and incubate in dark for 2 minutes before reading:
    • sgRNA 86: 66.9ng/ul
    • sgRNA 92: 17.4ng/ul
  • Total yeild in 39ul is:
    • 86: 39*66.9 = 2.6ug
    • 92: 39*17.4 = 678.6ng
  • Used online calculator to calculate the concentration in nM of each sgRNA. Used 100nt as the size (0.1kb)
    • 86: 1960nM
    • 92: 511.01nM

I need to make a 300nM solution for the digestion test, so this seems like it is a good amount. I am a little worried about my yield. The synthesis kit said it should produce 4-50ug of guide RNA. I am wondering if I should have used warmed water and a longer incubation time when eluting the RNA from the cleanup kit.