A | B | C | |
Material | Vendor | Catalog Number | |
Qubit 1X DS DNA High Sensitivity Assay Kit | Thermo Fischer Scientific (Waltham, MA, USA) | Q32851 | |
Alt-R® CRISPR-Cas9 tracrRNA, 20 nmol | Integrated DNA Technologies (Coralville, IA, USA) | 1072533 | |
Custom Alt-R® gRNA, 10 nmol | Integrated DNA Technologies (Coralville, IA, USA) | ||
HLS-CATCH Cassette Kit. | Sage Science (Beverly, MA, USA) | HIT0004 or HIT0012 | |
Hi-Bead HMW DNA Concentration Kit | Sage Science (Beverly, MA, USA) | HBK0012 | |
AMPure XP Bead-Based Reagent | Beckman Coulter Life Sciences (San Jose, CA, USA) | A63880 | |
KAPA HyperPlus Kit | Roche Holding AG (Basel, Switzerland) | KK8514 | |
Duplex Buffer | Integrated DNA Technologies (Coralville, IA, USA). | 11-04-02-01 | |
TaqMan™ Universal PCR Master Mix | Applied Biosystems by Thermo Fischer Scientific (Waltham, MA, USA) | 4364340 | |
StepOne Real-Time PCR System | Thermo Fischer Scientific (Waltham, MA, USA) | 4376357 | |
1.5 ml PCRclean tube DNA LoBind | Eppendorf (Hamburg, Germany) | 0030108051 | |
UltraPure DNase/RNase-Free Distilled Water | Invitrogen – Thermo Fischer Scientific (Waltham, MA, USA) | 10977-049 | |
0.75% agarose cassette | Sage Science (Beverly, MA, USA) | HIT0004 | |
NEB EnGen Spy Cas9 HF1, alternatively use wild-type Cas9 nuclease | New England Biolabs (Ipswich, MA, USA) | M0667M | |
TaqMan™ Copy Number Reference Assay, human, RNase P (VIC probe) | Thermo Fischer Scientific (Waltham, MA, USA) | 4403328 | |
Custom TaqMan™ Copy Number Assay (FAM probe) | Thermo Fischer Scientific (Waltham, MA, USA) | 4400296 |
A | B | C | D | |
Tube 1 | Tube 2 | |||
crRNA 1 | 4 µL | crRNA 2 | 4 µL | |
tracrRNA | 2.6 µL | tracrRNA | 2.6 µL | |
Duplex buffer | 19.4 µL | Duplex Buffer | 19.4 µL | |
Total Volume | 22 µL | Total Volume | 22 µL |
A | B | C | D | |
Tube 1 | Tube 2 | |||
crRNA 1 with tracr RNA (annealed) | 22 µL | crRNA 2 with tracr RNA (annealed) | 22 µL | |
4X enzyme buffer | 10 µL | 4X enzyme buffer | 10 µL | |
Cas9 (20 µM) | 8 µL | Cas9 (20 µM) | 8 µL | |
Total Volume | 40 µL | Total Volume | 40 µL |
A | B | C | |
Component | Volume per reaction | Volume total (for 25 reactions + 10%) | |
Master mix | 5µL | 137.5µL | |
bCD | 2µL | 55µL | |
probe | 0.5µL | 13.75µL | |
RNase P | 0.5µL | 13.75µL | |
Total | 8µL | 220µL |
A | B | |
Flow Cell Flush (FCF) | 1175µL | |
Flow Cell Tether (FCT) | 30µL | |
Total Volume | 1200µL |
A | B | |
DNA Library | 32µL | |
Sequencing buffer (SB) | 100µL | |
Library Beads (LIB) | 68µL | |
Total volume | 200 |
A | B | C | D | |
Tube 1 | Tube 2 | |||
crRNA 1 | 4 µL | crRNA 2 | 4 µL | |
tracrRNA | 2.6 µL | tracrRNA | 2.6 µL | |
Duplex buffer | 19.4 µL | Duplex Buffer | 19.4 µL | |
Total Volume | 22 µL | Total Volume | 22 µL |
A | B | C | D | |
Tube 1 | Tube 2 | |||
crRNA 1 with tracr RNA (annealed) | 22 µL | crRNA 2 with tracr RNA (annealed) | 22 µL | |
4X enzyme buffer | 10 µL | 4X enzyme buffer | 10 µL | |
Cas9 (20 µM) | 8 µL | Cas9 (20 µM) | 8 µL | |
Total Volume | 40 µL | Total Volume | 40 µL |
A | B | C | |
Component | Volume per reaction | Volume total (for 25 reactions + 10%) | |
Master mix | 5µL | 137.5µL | |
bCD | 2µL | 55µL | |
probe | 0.5µL | 13.75µL | |
RNase P | 0.5µL | 13.75µL | |
Total | 8µL | 220µL |
A | B | |
Flow Cell Flush (FCF) | 1175µL | |
Flow Cell Tether (FCT) | 30µL | |
Total Volume | 1200µL |
A | B | |
DNA Library | 32µL | |
Sequencing buffer (SB) | 100µL | |
Library Beads (LIB) | 68µL | |
Total volume | 200 |