Illumina Library Prep

The protocol for doing a library prep using Illumina kit.

Note: This protocol is unfinished

Background

Before you start this, you should determine the barcodes and index scheme for your library.

Then, you should draw an outline of what is happening at each step in this protocol:

Example Genomic Library Prep with inline barcodes and dual indexes

This protocol is based on the KAPA Hyper Prep Kit protocol with added notes from both Jon Puritz and Sara Schaal. We performed the protocol using half reactions of everything compared to the original protocol.

Kits used

KAPA Hyper Prep Kit (Kit Code: KK8505)

Materials needed

500 ng of DNA in 25 uL of RNAse free water that is sheared to the size appropriate for your library
Primers at 15 uM (aliquots of i5 and i7 custom primers if using)
Adapters at 15 uM (aliquots of annealed custom adapters if using)
KAPA beads (or other beads)

Equipment needed

Thermo Mixer set at 20C and 400 rpm (this can be done in the thermocycler as well if you don’t have a thermo Mixer)
Thermocycler - preprogrammed to have all thermocycler steps already set up: End Repair and A-Tailing and PCR.
Magnetic Bead Rack

Tips & Exposables

filtered 10 uL
filtered 20-200 uL
Strip tubes or individual tubes depending on how many libraries you are prepping

Protocol

End Repair & A-tailing

1.1 Assemble each end repair and A-tailing reaction in a tube or well of a PCR plate as follows:

Component	Volume
Fragmented Double Stranded DNA	25 uL
End Repair & A-tailing Buffer	3.5 uL
End Repair & A-tailing Enzyme	1.5 uL
Total Volume	30 uL

Depending on how many libraries you are prepping, you can make a master mix of the buffer and enzyme to add to each sample well. If you do this, be sure to add about 10% volume of each component due to pipetting error.

1.2 Vortex gently and spin down briefly. Return the plate/tube(s) to ice. Proceed immediately to the next step. 1.3 Incubate libraries in thermocycler set to the following protocol:

Step	Temp	Time
End Repair & A-Tailing	20 C	30 min
’’	65 C	30 min
HOLD	4 C	*

1.4 Proceed immediately to Adapter Ligation

Adapter Ligation

2.1 In the same plate/tube(s) in which end repair and A-tailing was performed, assemble each adapter ligation reaction as follows:

Component	Volume
End Repair & A-Tailing Reaction Product	30 uL
Adapter stock (concentration as required)	2.5 uL
PCR-grade water	2.5 uL
Ligation Buffer	15 uL
DNA Ligase	5 uL
Total Volume	55 uL

Again, at this step you can make a master mix of the buffer, Ligase enzyme and water. If you do this, be sure to add about 10% volume of each component due to pipetting error.

2.2 Mix thoroughly by pipetting up and down 15 times and centrifuge briefly. 2.3 Incubate at 20C for 60 minutes on the ThermoMixer with the rotations set at 400 rpm. 2.4 After incubation, proceed immediately to the next step

Post-Ligation Cleanup

3.1 In the same plate/tube(s), perform a 0.8X bead based cleanup by combining the following:

Component	Volume
Adapter ligation reaction product	55 uL
KAPA Pure Beads	44 uL
Total Volume	99 uL

3.2 Mix thoroughly by pipetting up and down 15 times. 3.3 Incubate the plate/tube(s) at room temperature for 10 min to bind DNA to the beads.