Generating Custom Sequencing Library

njh219 · 12-17-2014, 09:55 AM

Hi All,
Quick question regarding the orientation of the P5 and P7 regions when generating a sequencing library without using an Illumina kit.
When making forward and reverse primers to generate the library, should they be made so that the P5 and P7 regions are both on one strand, or with the complement of one on the same strand as the other.
example primer set A: 5'-P5-3' 5'-P7-3'
or
example primer set B: 5'-P5-3' 5'-Reverse_Complement_P7-3'
Obviously these primers will include additional elements; I'm not 100% clear on what the orientation of the P5 and P7 regions need to be.
Thanks.

njh219 · 12-17-2014, 11:52 AM

Hi all,
After looking into this more I figured out that it acts as in the second example (P5 and the complement of P7 are on the same strand).
Thanks.

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12-17-2014, 09:55 AM	#1
njh219 Junior Member Location: New York Join Date: Aug 2013 Posts: 8	Generating Custom Sequencing Library Hi All, Quick question regarding the orientation of the P5 and P7 regions when generating a sequencing library without using an Illumina kit. When making forward and reverse primers to generate the library, should they be made so that the P5 and P7 regions are both on one strand, or with the complement of one on the same strand as the other. example primer set A: 5'-P5-3' 5'-P7-3' or example primer set B: 5'-P5-3' 5'-Reverse_Complement_P7-3' Obviously these primers will include additional elements; I'm not 100% clear on what the orientation of the P5 and P7 regions need to be. Thanks.

12-17-2014, 11:52 AM	#2
njh219 Junior Member Location: New York Join Date: Aug 2013 Posts: 8	Hi all, After looking into this more I figured out that it acts as in the second example (P5 and the complement of P7 are on the same strand). Thanks.