Testing 77kb Guide RNAs 86 and 92 on the 77-2 PCR Product in a Cas9 Test Digestion

Dilutions and Preparation

  • sgRNAs were already diluted in previous digestion to 300nM
  • To make sure that the Cas9 was as enzymatically active, I did another dilution to 1uM:
    • 39ul diluent B
    • 2ul 20uM Cas9 enzyme
  • I had done 2 more 77-2 50ul PCR reactions, combined them together, and cleaned them up with beads
  • This yielded me not very much DNA still, but I decided to procede anyways
  • 77-2 PCR product was 14.4ng/ul
  • The size is 1,891bp
  • Equation for DNA ng/ul to nM:
    • nM = ((ng/ul)/(660g/mol * size in bp)) * 1000000
  • 77-2: ((14.4ng/ul)/(660g/mol * 1,891bp) * 1000000) = 12nM
  • I needed 30uM, but I decided to try it anyways, and to add an extra ul in to increase the amount of DNA

Set up and Procedure

tube number PCR product sgRNA Cas9 reaction type
1 77-2 No sgRNA No Cas9 control
2 77-2 No sgNRA + Cas9 control
3 77-2 +sgRNA 86 No Cas9 control
4 77-2 +sgRNA 92 No Cas9 control
5 77-2 +sgRNA 86 + Cas9 digest test
6 77-2 +sgRNA 92 + Cas9 digest test
  • Set the thermocycler program:
    • 25 decree C hold
    • 25 degree C 10 minutes
    • 37 degree C hold
    • 37 degree C 15 minutes
  • Thawed all reagents on ice and made mixes on ice
  • Made master mix:
    • 20ul molec grade water * 7 = 140ul
    • 3ul NEB buffer r3.1 * 7 = 21ul
  • Pipette mixed and spun down
  • Added 23ul master mix to each tube (of 6 strip tubes)
  • Added 3ul 300nM sgRNA 86 to tubes 3 and 5
  • Added 3ul 300nM sgRNA 92 to tubes 4 and 6
  • Added 3ul water to tubes 1 and 2
  • Added 1ul 1nM Cas9 to tubes 2, 5, and 6
  • Added 1ul water to tubes 1, 3, and 4
  • Pipette mixed tubes and spun them down
  • Incubated the tubes in the thermocycler for 10 minutes at 25 degrees C
  • Took out the tubes and added 4ul of 77-2 PCR to every tube
  • Pipette mixed and spun down tubes
  • Incubated tubes in the thermocycler for 15 minutes at 37 degrees C
  • Took tubes out and added 1ul of Qiagen proteinase K to each
  • Pipette mixed and spun down tubes
  • Incubated the tubes at room temp for 10 minutes
  • Afterwards, I put the tubes in the freezer overnight

Gel 20220525

  • I made a 2% gel for this
  • 10ul of sample were run with 2ul of loading dye
  • Gel ran at 100V for ~35 minutes
  • And stained for 35 minutes

This is a great gel pic, and it shows clear digestion for both sgRNAs, with 86 working better. I can see that it looks like sgRNA 92 is a little bit degraded/fuzzy or maybe lower concentration. That could explain that it has incomplete digestion.

It is great that this still worked even though the PCR concentration was not right.