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Old 07-11-2014, 06:28 AM   #1
Ahmed_BH
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Exclamation Possibility of tagmentation without fragmentation with Nextera XT kit

Hello everyone,

I would like to know if it's possible to do the fragmentation of my cDNA amplicon without any fragmentation using the Nextera XT kit. PCR amplicons have already sizes between 150 and 600 bp. these PCR products will be sequenced with Miseq.

Thanks in advance

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Old 07-11-2014, 06:45 AM   #2
Olaf Blue
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Hello Ahmed_BH,

Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base scission/cut at the site, which after the PCR to add adapters, generates a 9-base duplication at each insertion site. but the scission/fragmentation is a primary characteristic of Transposome insertion.
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Old 07-11-2014, 06:58 AM   #3
Ahmed_BH
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Question

Quote:
Originally Posted by Olaf Blue View Post
Hello Ahmed_BH,

Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base scission/cut at the site, which after the PCR to add adapters, generates a 9-base duplication at each insertion site. but the scission/fragmentation is a primary characteristic of Transposome insertion.
thanks for answers
so you think that it's possible to use any kit which use fragmentation/tagmentation step.
the particularity of my PCR products that i have ligated a specific index for everyone sample and then i have pooled all the samples. i want now to keep these index during the preparation of libraries for Miseq sequencing.
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Old 07-11-2014, 07:11 AM   #4
Olaf Blue
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The Nextera technology is unique. If you have PCR products that have ligated adapters/indexes, a tagmentation reaction will fragment the products and depending on the length of the PCR products, may well lose one of the adapter ends, which will be replaced by an adapter from the Nextera process.
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nextera sample prep, no fragme, pcr size amplicon, tation

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