The Illumina DNA Prep (previously Nextera DNA Flex) protocol is a versatile and efficient approach for producing high-quality sequencing libraries from DNA samples. This protocol utilizes bead-linked tagmentation technology, which integrates DNA fragmentation and adapter tagging into a single enzymatic reaction. The kit offers flexibility in accommodating a broad range of DNA inputs. If the total input falls below 100 ng, it is necessary to quantify and normalize the input DNA. However, if the input DNA quantity is between 100-500 ng, normalization is not required.
This protocol has been adapted from the Illumina DNA Prep protocol, which can be found here (full protocol) and here (checklist protocol). We also have a protocol for a low-cost variation called HackFlex, found here.
Illumina DNA Prep Kit
IDT for Illumina DNA/RNA UD Indexes Set A
IDT for Illumina DNA/RNA UD Indexes Set B
IDT for Illumina DNA/RNA UD Indexes Set C
IDT for Illumina DNA/RNA UD Indexes Set D
80% Ethanol, freshly prepared
Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting.
Bring to room temperature. Vortex to mix.
If precipitates are observed, heat at 37°C for 10 minutes and then vortex until dissolved.
Use at room temperature
Thaw on ice. Invert to mix, then briefly centrifuge.
Thaw at room temperature. Spin briefly before use.
Thaw and bring to room temperature. Vortex to mix.
Vortex and invert to mix.
Amplify Tagmented DNA
x # Samples
- 68°C for 3 min
- 98°C for 3 min
- X cycles of (see table below):
- 98°C for 45 sec
- 62°C for 30 sec
- 68°C for 2 min
- 68°C for 1 min
- 10°C hold
Clean Up Libraries
- 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 require individual quantification.
- Use the HS DNA 5000 or HS DNA 1000 tape and the appropriate reagent buffer. A successful library preparation will have a peak around 500 bp. See below for example traces.
- Quantify the sample with the HS DNA Qubit kit or PicoGreen.
Standard TapeStation Trace