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56 steps · 37 materials & instruments
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| # | Action | Timing |
|---|---|---|
| 1 | Turn on Covaris M220 focused-ultrasonicator and pre-cool to 4°C | 2 h |
| 2 | Transfer 20–50 μg DNA to MicroTUBE-130 and adjust volume to 130 μL with nuclease-free water | 2 h |
| 3 | Set Covaris M220 parameters: Temperature 4°C, Peak Power 50 W, Duty Factor 20%, Treatment Time 50–65 sec, Cycles 200 | 2 h |
| 4 | Fragment DNA to target peak at 300–700 bp by sonication | 2 h |
| 5 | Run 1 μL sonicated DNA on 1% (wt/vol) agarose gel in 1× TAE buffer to verify size distribution | 2 h |
| 6 | Prepare primer anneal mixture on ice: 16 μL 10× Bst buffer, 4 μL Bio-primer-MYC1 (+) (1 μM), 32 μL 5 M Betaine, 1–40 μg sonicated DNA, nuclease-free water to 160 μL | 1.5 h |
| 7 | Aliquot primer anneal mixtures into 4 PCR tubes | 1.5 h |
| 8 | Perform primer anneal: 95°C 3 min, then 5 cycles (95°C 2 min, 58°C 3 min), final 58°C 3 min, hold 10°C | 1.5 h |
| 9 | Prepare primer extension mixture: 4 μL 10× Bst buffer, 4 μL dNTPs (2.5 mM each), 2.5 μL Bst 3.0 DNA polymerase (8 U/μL), 29.5 μL ddH2O | 1.5 h |
| 10 | Add 10 μL primer extension mixture to each PCR tube and mix by vortex | 1.5 h |
| 11 | Perform primer extension: 65°C 15 min, 80°C 5 min (inactivation), hold 25°C | 1.5 h |
| 12 | Place AMPure XP beads at room temperature for at least 30 min before use | 40 min |
| 13 | Add 50 μL pre-warmed AMPure XP beads to each PCR tube, pipette up/down 15–20 times, incubate at room temperature for 5 min | 40 min |
| 14 | Place PCR tubes on magnetic stand (DynaMa-PCR Magnet) at room temperature for 5 min | 40 min |
| 15 | Remove and discard supernatant completely | 40 min |
| 16 | Add 200 μL 70% ethanol, remove supernatant completely (repeat twice) | 40 min |
| 17 | Add 50 μL 10 mM Tris-HCl, pH 7.5, pipette up/down 15–20 times, incubate 2 min | 40 min |
| 18 | Place on magnetic stand 2 min, transfer 50 μL supernatant to new 1.5 mL tube, pool same samples together | 40 min |
| 19 | Check concentration with spectrophotometer to confirm recovery | 40 min |
| 20 | Incubate purified DNA at 95°C for 5 min and immediately chill on ice for 3 min | 5 h |
| 21 | Prepare streptavidin binding mixture: 200 μL denatured DNA, 50 μL 5 M NaCl, 2.5 μL 0.5 M EDTA, 0.5 μL 10% Triton X-100 (total 253 μL) | 5 h |
| 22 | Transfer 20 μL streptavidin C1 beads to new 1.5 mL tube | 5 h |
| 23 | Add 400 μL 1× B&W buffer, mix by pipetting, place on DynaMag-2 1 min, remove supernatant (repeat twice) | 5 h |
| 24 | Suspend C1 beads with 20 μL 1× B&W buffer | 5 h |
| 25 | Add 20 μL washed streptavidin C1 beads to binding mixture and incubate on rotator at room temperature for 4 h | 4 h |
| 26 | Spin mixture at 200 g for 5 s (must be <3000 rpm) | 5 h |
| 27 | Place on DynaMag-2 holder 1 min, remove supernatant completely | 5 h |
| 28 | Wash C1 beads with 400 μL 1× B&W buffer, capture on magnet 1 min, discard supernatant (repeat twice) | 5 h |
| 29 | Resuspend beads with 400 μL 10 mM NaOH, immediately place on magnet 1 min, discard supernatant (incubation <2 min) | 5 h |
| 30 | Wash beads with 400 μL 10 mM Tris-HCl pH 7.5, capture on magnet 1 min, discard supernatant (repeat twice) | 5 h |
| 31 | Resuspend C1 beads with 42.4 μL 10 mM Tris-HCl, pH 7.5 | 5 h |
| 32 | Prepare bridge adapter mixture: 20 μL Bridge adapter-upper (400 μM) + 20 μL Bridge adapter-lower (400 μM) | 5 h |
| 33 | Anneal bridge adapter: 95°C 3 min, then ramp 85°C to 30°C at 0.1°C/s with 1 min holds, hold 10°C | 5 h |
| 34 | Add 120 μL nuclease-free water to bring concentration to 50 μM, prepare 80 μL aliquots, store at -20°C | 5 h |
| 35 | Set up 80 μL ligation: 42.4 μL ssDNA on C1 beads, 8 μL 10× T4 ligase buffer, 1.6 μL Bridge adapter (50 μM), 4 μL T4 DNA ligase (400 U/μL), 24 μL 50% PEG8000 | 5 h |
| 36 | Mix well (except PEG8000), add PEG8000 with cut-off P200 tip, mix by pipetting 10–15 times | 5 h |
| 37 | Incubate ligation reaction at room temperature for 4 h or overnight (12–16 h) on rotator at 8 rpm | 4 h or overnight |
| 38 | Add 320 μL 1× B&W buffer, capture beads on magnet 1 min, remove supernatant | 5 h |
| 39 | Wash beads with 400 μL 1× B&W buffer (repeat twice), then 10 mM Tris-HCl pH 7.5 (repeat twice) | 5 h |
| 40 | Resuspend on-beads ligated DNA with 73 μL 10 mM Tris-HCl, pH 7.5 | 5 h |
| 41 | Set up 100 μL nested PCR: 73 μL on-beads ligation products, 10 μL 10× EasyTaq buffer, 8 μL dNTPs (2.5 mM), 4 μL I5-Nested-1-MYC1 (+) (10 μM), 4 μL I7-index primer (10 μM), 1 μL EasyTaq DNA polymerase (5 U/μL) | 1.5 h |
| 42 | Aliquot nested PCR mixture into 2 PCR tubes and perform: 95°C 5 min, then 15 cycles (95°C 1 min, 58°C 45 s, 72°C 1 min), 72°C 5 min, hold 10°C | 1.5 h |
| 43 | Place AMPure XP beads at room temperature for at least 30 min | 40 min |
| 44 | Add 40 μL pre-warmed AMPure XP beads to each PCR tube, pipette 15–20 times, incubate 5 min | 40 min |
| 45 | Place on magnetic stand 5 min, remove supernatant, wash with 200 μL 70% ethanol twice | 40 min |
| 46 | Add 35 μL 10 mM Tris-HCl pH 7.5, incubate 2 min, transfer 33 μL supernatant to 1.5 mL tube, pool samples | 40 min |
| 47 | Check concentration with spectrophotometer (should be 1–3 ng/μL) | 40 min |
| 48 | Set up 100 μL tagged PCR: 63 μL PCR products, 20 μL 5× FastPfu buffer, 8 μL dNTPs (2.5 mM), 4 μL P5-I5 (10 μM), 4 μL P7-tag (10 μM), 1 μL FastPfu DNA polymerase (2.5 U/μL) | 1 h |
| 49 | Aliquot into 2 PCR tubes, perform: 95°C 2 min, then 10–15 cycles (95°C 30 s, 58°C 45 s, 72°C 1 min), 72°C 5 min, hold 10°C | 1 h |
| 50 | Pool same samples, add 20 μL 6× DNA loading buffer, run on 1.5% agarose gel in 1× TAE buffer | 3 h |
| 51 | Excise gel with DNA fragments 300–700 bp, cut into small pieces, transfer to 15 mL tube | 3 h |
| 52 | Add 2 mL binding buffer, incubate at 56°C until gel dissolved | 3 h |
| 53 | Transfer through column at 10,000 g for 1 min, discard flow-through | 3 h |
| 54 | Add 800 μL wash buffer, spin at 10,000 g for 1 min, discard flow-through | 3 h |
| 55 | Spin at 10,000 g for 2 min, transfer to new 1.5 mL microtube | 3 h |
| 56 | Add 50 μL nuclease-free water, incubate 2 min, spin at 10,000 g for 2 min, collect eluted fraction | 3 h |
| Name | Vendor | Catalog |
|---|---|---|
| Nuclease-free water | Milli-Q,0.22mmfiltered | N/A |
| 1 M Tris-HCl, pH 7.5 | Invitrogen | Cat#15567027 |
| 5 M NaCl | Sigma-Aldrich | Cat#S6546 |
| 0.5 M EDTA, pH 8.0 | Invitrogen | Cat#15575020 |
| Ethanol | Sigma-Aldrich | Cat#M3148 |
| Bst 3.0 DNA Polymerase | NewEnglandBioLabs | Cat#M0374L |
| dNTPs, 2.5 mM each | TransGenBiotech | Cat#AD101-01 |
| Betaine solution, 5M | Sigma-Aldrich | Cat#B0300 |
| Triton X-100 | Sigma-Aldrich | Cat#T8787 |
| 10× T4 DNA ligase buffer | ThermoScientific | Cat#EL0011 |
| T4 DNA ligase | ThermoScientific | Cat#EL0011 |
| PEG8000 | Sigma-Aldrich | Cat#89510 |
| EasyTaq DNA Polymerase | TransGenBiotech | Cat#AP111-01 |
| AMPure XP | BeckmanCoulter | Cat#A63880 |
| FastPfu DNA Polymerase | TransGenBiotech | Cat#AP221-01 |
| Agarose | ThermoScientific | Cat#75510019 |
| Trans DNA Marker II | TransGenBiotech | Cat#BM411-01 |
| 6× DNA loading buffer | BeyotimeBiotech | Cat#D0072 |
| 50× TAE buffer | ThermoScientific | Cat#B49 |
| GeneJET Gel Extraction Kit | ThermoScientific | Cat#K0692 |
| Dynabeads MyOne streptavidin C1 beads | ||
| Bio-primer-MYC1 (+) | ||
| Bridge adapter-upper | ||
| Bridge adapter-lower | ||
| I5-Nested-1-MYC1 (+) | ||
| P5-I5 | ||
| P7-Tag | ||
| Centrifuge | Eppendorf | Cat#5406000291 |
| Spectrophotometer | DeNovix | Cat#DS-11FX+ |
| M220 Focused-ultrasonicator | Covaris | Cat#500295 |
| MicroTUBE-130 AFA Fiber | Covaris | Cat#520045 |
| PCR machine | ||
| DynaMag-2 | TransGenBiotech | Cat#BM411-01 |
| DynaMa-PCR Magnet | ||
| VWR tube rotator UK plug | ||
| 1.5 mL Microtubes | Covaris | Cat#520045 |
| 0.2 mL PCR strip tubes |
Direct package: protocol.json