Today, we are going to show you how to make
DNA libraries using the NEBNext Ultra II DNA
Library Prep Kit for Illumina.
Before you begin, you'll want to make sure that you have available all of the required
reagents and equipment that are not included in the kit.
Your DNA should be fragmented to the size range suitable for your sample and sequencing
requirements. This can be done using either mechanical methods or enzymatic methods.
The volume of your sample should be adjusted to 50 ul, and this should contain 500 pg to
1 ug of fragmented DNA.
Although the Ultra II kit can be used with very low input amounts, we recommend using
higher amounts if available, to minimize the number of PCR cycles required.
When working with the reagents included in the kit, it's important to
ensure that they are well mixed before use.
The first steps in library construction are End Repair and dA-Tailing. In this step,
the input DNA is blunted and phosphorylated, and an A is added to the 3’ end of each
fragment.
In the Ultra II workflow, these two steps are combined. You will be using the End
Prep Enzyme Mix and the End Prep Reaction Buffer, which are in the green-capped vials.
For each library, add 3 ul of the End Prep Enzyme Mix and 7 ul of the End Prep Reaction
Buffer to your 50 ul fragmented DNA sample.
Mix well by pipetting up and down 10 times.
Incubate this in a thermocycler at 20°C for 20 minutes, followed by 65°C for 20 minutes.
Then, hold at 4°C.
In this step, adaptors with a single T overhang are ligated to the end-repaired, dA-tailed fragments.
If you started with less than 100 ng of input DNA, we recommend using lower amounts
of adaptor to reduce the risk of adaptor-dimer formation.
The reagents used in this step are the Ultra II Ligation Master Mix, the Ligation
Enhancer, and the Adaptor, which are in the red-capped vials.
Note that the Ultra II Ligation Master Mix is very viscous, so it's important to
pipette slowly to ensure you are getting the desired volume.
The Ultra II Ligation Master Mix and Ligation Enhancer can be combined ahead of time and
stored at 4°C and will be stable for at least 8 hours.
Add the Ultra II Ligation Master Mix, Ligation Enhancer, and Adaptor to the End
Prep reaction, and mix well. We recommend setting a 100 or 200 ul pipette to 80 ul,
and then pipetting up and down at least 10 times. A quick spin will collect all liquid
from the sides of the tube.
Incubate at 20°C for 15 minutes in a thermocycler, with the heated lid turned off.
25. If you are using the NEBNext Adaptor, add 3 ul of USER enzyme and mix. Incubate
at 37°C for 15 minutes, with the heated lid set to at least 47°C.
If necessary, samples may now be stored at -20°C overnight.
Size selection enriches for molecules that were sheared to the desired size, and
have an adaptor ligated to each end.
Size selection is accomplished using magnetic beads. There are two rounds of selection:
the first removes DNA fragments larger than the desired size, and the second removes DNA
fragments smaller than the desired size. This is accomplished using specific ratios of the
bead solution to total volume, and the volume of beads required varies depending on the
desired fragment size.
Magnetic beads should be used at room temperature, and they should be vortexed before
use.
For the first round of size selection, add vortexed beads to the reaction and mix
well by pipetting up and down at least 10 times. Incubate for 5 minutes at room temperature,
then place the reaction into the magnetic field. After approximately 5 minutes the beads
should have separated from the supernatant.
Carefully remove the supernatant and avoid disturbing the pellet. Vertical bar magnets
pull the beads to one side of the tube or well, and the supernatant can be removed by
placing the pipette tip on the opposite side of the tube or well from the magnet. Plates
with circular magnets pull the beads uniformly to the walls of the tube or well, so the supernatant
can be removed by pipetting straight down into the bottom of the tube or well, being
careful not to touch the sides.
In this first round of size selection, the unwanted large fragments are bound to
the beads, and the desired DNA is in the supernatant. Retain the supernatant!
For the second round of size selection, add new magnetic beads, incubate for 5 minutes,
expose to a magnetic field, and remove the supernatant. Discard the supernatant, as the
desired DNA is bound to the beads in the pellet.
Wash the beads with 200 ul of 80% ethanol while they are in the magnetic field. Incubate
for 30 seconds at room temperature, and remove the ethanol. Repeat this wash step once.
Allow the beads to air dry while the beads are in the magnetic field, but avoid over-drying
the pellet.
Remove the tube or plate containing the dried bead pellet from the magnet, and elute
the library from the beads by adding 17 ul of Tris-HCl or 0.1X TE Buffer, followed by
mixing well by pipetting up and down or vortexing. A quick spin may be required to collect the
liquid to the bottom of the tube or plate. Incubate for an additional 2 minutes at room
temperature.
Return the sample to the magnet for ~5 minutes, or until the sample clears, and remove
15 ul of the supernatant. Add this 15 ul to a fresh PCR tube for library amplification.
The final step of library preparation is library amplification by PCR. This step
not only increases the amount of library, but also selects for moleculaes that have
an adaptor ligated to each end.
For multiplexed libraries, indices or barcodes can be introduced at this step, if
the NEBNext Adaptor and Primers are used.
To each sample, add the universal PCR primer, an index primer, and the Ultra II
Q5 Master Mix, which are in the blue-capped vials.
Mix thoroughly, as shown previously.
The number of PCR cycles will vary and is determined by the original
amount of input DNA.
This step also uses magnetic beads, but this time there is a single step, after which
the desired DNA – the library – is bound to the beads. Don't discard the beads!
Add 45 ul of vortexed magnetic beads to the reaction, and mix well by pipetting up
and down at least 10 times. Incubate at room temperature for 5 minutes, followed by exposure
to a magnetic field. After 5 minutes, remove and discard the supernatant, without disturbing
the bead pellet.
Wash the beads with freshly-prepared 80% ethanol, as previously demonstrated. Allow the
pellet to dry.
Remove the tube or plate containing the dried bead pellet from the magnet, and elute
the library from the beads by adding 33 ul of 0.1X TE Buffer, followed by mixing well.
After a quick spin, incubate at room temperature for 2 minutes.
Return the sample to the magnetic field until the sample clears, approximately 5 minutes,
and remove 30 ul of the supernatant – containing the library – to a new tube. This library
can be stored at -20°C.
You can confirm the size distribution of the library by diluting 1 ul of the library
5-fold with 10 mM Tris-HCl or 0.1X TE Buffer, and running it on the Bioanalyzer using the High-Sensitivity Chip.
The final library can be quantitated using qPCR-based methods, such as the NEBNext Library
Quant Kit, or using electrophoretic methods, like the Bioanalyzer.
For full details, see the manual for the NEBNext Ultra II DNA Library Prep Kit for
Illumina, which can be downloaded from our website. For any technical inquiries, please
contact NEB technical support.