Introduction
This protocol is a low-cost alternative to the Illumina DNA Prep protocol (previously Nextera DNA Flex). The paper describing Hackflex can be found here. Hack Flex is similar to the official protocol, but uses diluted Bead-Linked Transposomes and lab-made buffers.
The Illumina DNA Prep protocol can be found here.
Reagents
Reagent | Vendor | Catalog | Storage |
Illumina DNA Prep Kit | Illumina | 20060059 | Various |
IDT for Illumina DNA/RNA UD Indexes Set A | Illumina | 20027213 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set B | Illumina | 20027214 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set C | Illumina | 20042666 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set D | Illumina | 20042667 | -20°C |
80% Ethanol, freshly prepared | NA | NA | RT |
Molecular-Grade (Nuclease-Free) Water | Sigma | W4502-1L | RT |
Tris HCI Buffer, pH 7.6, 1 M | Crystalgen | 221-230 | 4°C |
MgCl2, 1 M | VWR | E525-100ML | RT |
N,N-DIMETHYLFORMAMIDE >99%, liquid | Sigma | D4551-250ML | RT, in flammable cabinet |
UltraPure 10% SDS | ThermoFisher | 15553027 | RT |
PrimeSTAR GXL DNA Polymerase Kit | Takara | R050A | -20°C |
Tris-EDTA (TE) Buffer | FisherScientific | BP2473-1 | RT |
SPRI (Homemade) or AmpureXP Beads | 4°C |
Plasticware | Vendor | Catalog |
96-well Hard Shell Plates (HSP) for Zephyr | REVVITY | 6008870 |
150 ul filter tips for Zephyr | REVVITY | 111426 |
StorPlate 450 ul V bottom plates for Zephyr | REVVITY | 6008290 |
2 ml Deep Well Plate (DWP) for Zephyr | REVVITY | 6008880 |
Lids for Zephyr | REVVITY | 6000030 |
12-Column Reservoir Seahorse DWP for Zephyr | REVVITY | 6008700 |
287 ml Reservoir for Zephyr | REVVITY | 6008730 |
Protocol
Prepare Reagents
Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting.
(Split between 2 50 mL conicals)
Bring to room temperature. Vortex to mix.
Reagent | [Stock] | [Final] | Volume (ml) |
Tris HCI Buffer, pH 7.6, 1 M | 1 M | 20 mM | 1 |
MgCl, 1 M | 1 M | 20 mM | 1 |
N,N-DIMETHYLFORMAMIDE >99%, liquid | >99% | 50% | 25 |
Molecular Grade Water | NA | NA | 23 |
Total | NA | NA | 50 |
If precipitates are observed, heat at 37°C for 10 minutes and then vortex until dissolved.
Reagent | [Stock] | [Final] | Volume (ml) |
Ultrapure SDS | 10% | 0.2% | 1 |
Molecular Grade Water | NA | NA | 49 |
Total | NA | NA | 50 |
Filter through 0.22 um filter before use. Use at room temperature
Reagent | [Stock] | [Final] | Amount |
PEG 8000 | Solid | 10% | 25 g |
NaCl | 5 M | 0.25 M | 12.5 ml |
Tris-EDTA (TE) buffer (10 mM Tris and 1 mM EDTA) | 1X | NA | to 250 ml |
Total | NA | NA | 250 ml |
Thaw at room temperature. Spin briefly before use.
Thaw and bring to room temperature. Vortex to mix.
Equilibrate to RT for at least 30 min. Vortex and invert to mix.
Dilute DNA
Normalization and Pooling | |||
Sample Well ID | Sample Volume | Diluent Volume | Destination Well ID |
A01 | 5 | 148 | A01 |
B01 | 5 | 76 | B01 |
C01 | 9 | 4.23 | C01 |
D01 | 5 | 19 | D01 |
E01 | 5 | 149.5 | E01 |
F01 | 5 | 156.5 | F01 |
⌠| ⌠| ⌠| ⌠|
(File â Open Application â Pooling and Normalization).
Item | Position | Plasticware | Contents |
150 ul Filter Tips | A2 | Box Filter Tips | Full Box |
DNA Source Plate | A3 | 96-well HSP | Extracted DNA (thawed, vortexed and spun down) |
DNA Destination Plate | B3 | 96-well HSP | Empty |
EB/Water | B4 | 287 ml Reservoir | EB or Water or C6 |
Run the âNextera Flex Tagmentation and PCR Setupâ Protocol on Zephyr
Run the âStandard Post-PCR Size Selectionâ Protocol on Zephyr
Next Steps
- Libraries with DNA Inputs of 100-500 ng generated in the same experiments do not require quantification individually. These can be pooled in a microcentrifuge tube. Add 5 ul of each library (up to 384), vortex to mix, and centrifuge briefly before quantifying the pool.
- Libraries with DNA Inputs of <100 ng (i.e. HackFlex libraries!) require individual quantification.
- Use the HS DNA 5000 tape and the appropriate reagent buffer and ladder. A successful library preparation will have a peak of around 500 bp. See below for example traces. Calculate the average fragment size of the individual libraries.
- Quantify the libraries with the Picogreen.
- Use the concentration and average fragment size to calculate the molarity of individual samples.
- Pool equal molar concentrations of each library.
- Calculate pool molarity by measuring concentration via Qubit and average fragment size via TapeStation.
- Dilute to 2 nM (NextSeq) or 1 nM (MiniSeq) before preparing the run
HackFlex Tapestation Trace