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Old 05-07-2014, 04:46 PM   #1
Ayaka
Junior Member
 
Location: southamerica

Join Date: Apr 2014
Posts: 6
Question Newbie:Use of kit for library prep.Why can I just amplify using index,adaptor seq.?

Hi there, I am very new in the sequencing world and I am gathering the needed info to start the firs sequencing project of 16S amplicons with MiSeq in our lab.
My question is regarding the library prep. I donīt understand why there is the need to use kits like Nextera or Truseq to attach the index or adaptors needed for pooling and sequencing with the illumina platforms, if those sequences are available. Could I just make those as primers and perform a PCR? without the need of the kit? if so, what is the point of using the kit?

I have an illumina manual were they sequence 16S amplicons and use the nextera kit and perform 2 PCR reaction. Why does it has to be done in 2 PCR reaction, instead of just 1? is it because of the Tm of the primer would be not adequate?

I am sure these are very simple questions! but I havenīt been able to find satisfying answers. I hope someone can shed some light into my querys.
Thank you in advance

Last edited by Ayaka; 05-07-2014 at 04:53 PM. Reason: I added one more query
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