Tag Archives: CleanTag

What’s the difference between the CleanTag™ Ligation Kit and CleanTag™ Library Prep Kit?

Posted on by

Both products included the reagents needed for the ligation of the 3′ adapter and 5 ‘ adapter. The CleanTag™ Small RNA Library Prep Kit (Cat# L-3206) also includes the reagents needed for the RT and PCR steps of library preparation which were not included in the CleanTag™ Ligation Kit (Cat# L-3203).

CleanTag™ Small RNA Library Prep Kit
(Cat# L-3206)		 CleanTag™ Ligation Kit
(Cat# L-3203)
CleanTag™ 3′ Adapter CleanTag™ 3′ Adapter
CleanTag™ 5′ Adapter CleanTag™ 5′ Adapter
Enzymes, 1 & 2 Enzymes, 1 & 2
Buffers, 1 & 2 Buffers, 1 & 2
Reverse Transcriptase
RT Buffer
dNTP Mix (10 mM ea)
DTT
High Fidelity PCR Master Mix (2X)
RNase Inhibitor RNase Inhibitor

The CleanTag™ Ligation Kit was discontinued in early 2016.

Which “High Fidelity PCR Master Mix” would you recommend using with the CleanTag Ligation Kit for Small RNA Library Prep? I noticed your protocol uses more than the volume that comes with the TruSeq Small RNA kit, so I was wondering which PCR MasterMix have you found most success with? -Steve

Posted on by

Hi Steve,

Thank you for your question. We recommend the Q5® High Fidelity 2x Master Mix (catalog # M0492S) from NEB for use with the CleanTag™ Ligation Kit for Small RNA Library Prep. You can view our full list of recommended reagents here.

Please let me know if you have any other questions.

Best regards,
Elizabeth

I just used your CleanTag Ligation Kit for Small RNA and I am trying to determine if the ligation/library prep was successful. Am I correct in assuming that I expect to see essentially no adaptor dimer formation in the prep? The TapeStation results show large (~2-4 ng/µl) peaks at around ~140bp for each sample (magnetic bead cleaned) and no obvious peaks at what would be the adaptor-dimer (~120bp). I just wanted to confirm that because of the CleanTag chemistry, no adaptor-dimers form during ligation, and therefore, they aren’t amplified. Is this correct? If true, I should also be able to load these preps straight onto a sequencer with no gel purification of the 140bp band? – Steve

Posted on by

Dear Steve,

You are correct in that the CleanTag™ chemistry can fully suppress adapter dimer formation. If you are using very low input levels (less than 10 ng) you may start to see some adapter dimer but it should be minimal compared to amount of tagged library. Dilution of the adapters per the product insert is key to keep adapter dimer low at lower inputs.

Yes, you can load magnetic bead purified samples without gel purification directly onto a sequencer and get good quality sequencing data.

Best regards,
Sabrina Shore, MS
Scientist