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Old 09-03-2014, 07:55 AM   #1
RNAarecrazy
Junior Member
 
Location: Massachusettes

Join Date: Jan 2014
Posts: 4
Default Reply from NEB re: ssDNA library prep

I thought this might be helpful for some of you.

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Thank you for contacting NEB Technical Support.


Dear Chris,

Your question and answer are below:

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

Hi NEB,
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



Answer:
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.
Deyra
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