Whole genome amplification: In PCR workstation, add 1 µl exo-resistant random primers to PCR tube with single cell.
{"action": "add", "output": "", "reagent": ["exo-resistant"], "volume": ["1 µl"]}
keep on ice for 10 min #####%%%%% output "µl stop buffer (400 mM HCl, 600 mM Tris-HCl pH 7.5)" are used in line6
{"action": "keep", "output": "\u00b5l stop buffer (400 mM HCl, 600 mM Tris-HCl pH 7.5)", "temperature": ["ice"], "time": ["10 min"], "reagent": [""]}
Add 3 µl stop buffer (400 mM HCl, 600 mM Tris-HCl pH 7.5) to neutralize lysis buffer, .
{"action": "add", "output": "", "reagent": ["\u00b5l stop buffer (400 mM HCl, 600 mM Tris-HCl pH 7.5)"], "volume": ["3 µl"]}
keep on ice for 2 min #####%%%%% output "2 µl Phi29 polymerase" are used in line10
{"action": "keep", "output": "2 \u00b5l Phi29 polymerase", "temperature": ["ice"], "time": ["2 min"], "reagent": [""]}
Add 32 µl master mix (30 µl MDA reaction buffer, 2 µl Phi29 polymerase), mix gently.
{"action": "combine", "output": "", "volume": ["32 \u00b5l master mix 30 \u00b5l MDA reaction buffer,"], "reagent": ["2 \u00b5l Phi29 polymerase"]}
Remove clear liquid, . #####%%%%% output "clear liquid" are used in line22
{"action": "remove", "output": "clear liquid", "reagent": [""]}
wash beads twice with fresh 80% ethanol
{"action": "rinse", "output": "", "reagent": ["beads"]}
Air-dry beads for about 5 min, avoid cracking. #####%%%%% output "nuclease-free water" are used in line18
{"action": "minimize", "output": "nuclease-free water", "length": [""]}
Elute beads with 32 µl nuclease-free water, .
{"action": "elute", "output": "", "reagent": ["nuclease-free water"], "volume": ["32 \u00b5l"]}
use magnetic separator until clear,
{"action": "use", "output": "", "device": ["magnetic separator"]}
transfer 30 µl to clean tube
{"action": "transfer", "output": "", "volume": ["30 \u00b5l"], "container": ["tube"], "reagent": ["clear liquid"]}
Concentration measurement: Dilute purified product 10-20 times, quantify with Qubit (yield 10-12 µg). #####%%%%% output "TBE," are used in line26
{"action": "quantify", "output": "TBE,", "mass": ["10-12 µg"], "reagent": ["", ""]}
Check product size by agarose gel electrophoresis (1.0% agarose gel, 1.0% TBE, 100V, 40 min). #####%%%%% output "product size" are used in line28
{"action": "check", "output": "product size", "device": ["agarose gel electrophoresis"]}
Prepare 96-well plate with PCR mixture: 5 µl Fast Sybr green master mix, .
{"action": "prepare", "output": "", "container": ["96-well plate"], "reagent": ["PCR mixture", "product size"]}
Seal plate, spin down, incubate: 95°C for 20s, .
{"action": "incubate", "output": "", "device": ["PCR machine"], "temperature": ["95°C"], "time": ["20s"]}
Calculate relative uniformity values (RUVs) for 8 loci, select samples with RUV values for at least 6 of 8 loci between 0.25-4 for library construction. #####%%%%% output "relative uniformity values (RUVs)" are not used
{"action": "calculate", "output": "relative uniformity values (RUVs)"}