FAQs on the Illumina DNA Prep, (M) Tagmentation Library Preparation protocol

How long does it take to generate ready-to-sequence Illumina DNA Prep libraries?

• It takes 2 to 3 hours to prepare libraries; the time is dependent on the number of libraries that are prepared.

How do to choose index combinations for libraries when pooling a small number of samples?

• See the Index Adapter Pooling Guide.

What is the purpose of double-sided bead purification after PCR?

• The purpose of the double-sided bead purification is to size select the library fragments; the two-step process first removes the large fragments and the second step removes the small molecular weight fragments.

What causes sample frothing and is this detrimental to library preparation?

• Samples froth because of a detergent used in the library prep. As long as the wash step is included (as described in the protocol), the libraries will not be affected. If desired, gentle pipetting up and down will reduce the amount of froth.

What happens if the BLT beads dry?

• This will negatively impact the library preparation; do not allow the BLT to dry.

Are the index sequences publicly available?

• Yes, they are available in BaseSpace Sequence Hub (BSSH) and the Illumina Adapter Sequences Document.

How to proceed if BLT have different visual appearance after washing?

• Proceed as long as beads are clearing after incubation on the magnet.

It is okay if some of the BLT are carried forward after the PCR?

• Visually inspect and re-clear the supernatant on the magnet if BLT carried over.

Can the BLT (and reaction mix) withstand centrifugation?

• Yes. For spinning down, use a low speed for a few seconds if needed, and resuspend BLT by pipetting.

How to proceed if too much or too little BLT reagent was added?

• This is not recommended or supported. Results are not guaranteed, libraries may still be generated but the yield may be lower and normalization of library yields may not be achieved.

How many freeze thaw cycles are supported for the library preparation reagents?

• Illumina has tested frozen reagents up to four (4) freeze/thaws of frozen reagents without observing any loss of activity.

How long can the tagmentation reaction remain at 10°C hold after the 55°C for 15 mins?

• This is not a safe stopping point, proceed with the protocol.

For any feedback or questions regarding this article (Illumina Knowledge Article #1260), contact Illumina Technical Support [e-mail].