Indexed PCR Primer Design

ksil91 · 12-01-2014, 07:40 AM

Hello, I'm new here so apologies if this question has been answered.

I'm following the Buckler Lab GBS protocol and only have access to 48 adapters for ApeKI. I'd like to multiplex these into one Illumina lane using indexed PCR primers. I found sequences for PCR primers from the ddRAD protocol(Peterson). Do I have to alter the sequence at all to match my ApeKI adapters? Do I have to request anything particular when ordering these primers from IDT? Or would it be better for me to just order the NEBNext Multiplex Oligos for Illumina Kit? (https://www.neb.com/products/e7335-n...-primers-set-1)

ApeKI Common Adapter:
5'-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

ApeKI Barcoded Adapter:
5’-CWGxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTyyyy

PCR primers I'm considering ordering:
PCR1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
PCR2_Idx_1_ATCACG: CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_2_CGATGT: CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_3_TTAGGC: CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_4_TGACCA: CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC

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12-01-2014, 07:40 AM	#1
ksil91 Junior Member Location: Chicago, IL Join Date: Nov 2014 Posts: 2	Indexed PCR Primer Design Hello, I'm new here so apologies if this question has been answered. I'm following the Buckler Lab GBS protocol and only have access to 48 adapters for ApeKI. I'd like to multiplex these into one Illumina lane using indexed PCR primers. I found sequences for PCR primers from the ddRAD protocol(Peterson). Do I have to alter the sequence at all to match my ApeKI adapters? Do I have to request anything particular when ordering these primers from IDT? Or would it be better for me to just order the NEBNext Multiplex Oligos for Illumina Kit? (https://www.neb.com/products/e7335-n...-primers-set-1) ApeKI Common Adapter: 5'-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT ApeKI Barcoded Adapter: 5’-CWGxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTyyyy PCR primers I'm considering ordering: PCR1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG PCR2_Idx_1_ATCACG: CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGC PCR2_Idx_2_CGATGT: CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC PCR2_Idx_3_TTAGGC: CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGC PCR2_Idx_4_TGACCA: CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC