Smartseq Exonuclease

wyue · 10-15-2019, 09:35 AM

Hi all,

We have a few questions about using exonuclease before the preamp step of the Smartseq protocol.

1. Is this step intended for removing primer dimers, not excess single-stranded primers? Is that why lambda exonuclease (which works on dsDNA) is chosen instead of ExoI?

2. Wouldn't lambda exonuclease digest away the double-stranded cDNA template? If the ends got digested, the priming sites may be lost.

3. We have tried replacing lambda exonuclease with ExoI for a 30 min incubation, then an 85C, 15 min inactivation before adding primers for preamp step. However, yield is very low. Can somebody explain why?

4. Does anyone have the reference for the first suggestion of using lambda exonuclease?

Many thanks!

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Cel Seq2 Exonuclease and cell barcodes	StephanK	Sample Prep / Library Generation	0	12-09-2016 05:00 AM
SmartSeq cDNA validation with TapeStation	vtorda	Sample Prep / Library Generation	1	11-23-2016 12:50 PM
SMARTseq mutations	Bowser	Sample Prep / Library Generation	0	11-18-2016 02:23 AM
Exonuclease I	airun78	Sample Prep / Library Generation	1	07-18-2014 10:29 AM
Fully exonuclease-resistant custom oligonucleotides?	memeri	Sample Prep / Library Generation	5	10-31-2011 08:43 AM

10-15-2019, 09:35 AM	#1
wyue Junior Member Location: San Francisco Join Date: Oct 2019 Posts: 1	Smartseq Exonuclease Hi all, We have a few questions about using exonuclease before the preamp step of the Smartseq protocol. 1. Is this step intended for removing primer dimers, not excess single-stranded primers? Is that why lambda exonuclease (which works on dsDNA) is chosen instead of ExoI? 2. Wouldn't lambda exonuclease digest away the double-stranded cDNA template? If the ends got digested, the priming sites may be lost. 3. We have tried replacing lambda exonuclease with ExoI for a 30 min incubation, then an 85C, 15 min inactivation before adding primers for preamp step. However, yield is very low. Can somebody explain why? 4. Does anyone have the reference for the first suggestion of using lambda exonuclease? Many thanks!