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Old 09-03-2014, 08:55 AM   #1
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Location: Massachusettes

Join Date: Jan 2014
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Default Reply from NEB re: ssDNA library prep

I thought this might be helpful for some of you.

Thank you for contacting NEB Technical Support.

Dear Chris,

Your question and answer are below:

Sent: Thursday, August 28, 2014 3:28 PM
To: Info
Subject: NEBNext RNA Library kit for ssDNA?

Can I adapt the NEBNext RNA library to generate library using ssDNA as starting material? I assume I can just start the protocol beginning from the second strand cDNA synthesis. Correct me if I am wrong..
Thank you

In the NEBNext RNA library prep kits, the second strand cDNA synthesis has been optimized to generate a second strand cDNA from the DNA-RNA hybrid resulting from the first strand synthesis step.

During second strand synthesis, RNA primers are generated by the RNase H enzyme present in the second strand synthesis enzyme mix. RNase H nicks the RNA template, creates RNA primers. This primers are extended by DNA Pol I present in the enzyme mix. Finally, E.coli DNase Ligase close the nicks.

In your case, since you already have ssDNA, I would suggest to make double stranded DNA using random primers. Then take that DNA into the E7370 - NEBNext® Ultra™ DNA Library Prep Kit for Illumina®.

I hope this is helpful. Please contact us back if you have further questions.
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Old 12-23-2019, 11:07 PM   #2
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Technical support system is now here for helping you. With more helping purpose where you get technica support are always helping you more and with all of the given strategies where you get work on website. I am here to do different work to support the whole technical support system.
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