Brandon:LabNotes/Project1/2012-5-8
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Nextera modification, Adding T7
- T7 would be added by PCR after tagmentation.
- Should result in less sample loss since adapters will be designed for both adaptor 1 and adaptor 2
- Efficiency will be high in terms of capture of insertions, but not 100% since PCR is not 100% efficient
Current nextera sequences 5'-G[CCTCCCTCGCGCCATCAG][AGATGTGTATAAGAGACAG]-3' Blue ME 5'-G[CCTTGCCAGCCCGCTCAG][AGATGTGTATAAGAGACAG]-3' Orange ME Assumed nextera transposons for transposon loading or before restriction digest after loading. Blue 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' 3'- TCTACACATATTCTCTGTC/5phos/ -5' Orange 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3' 3'- TCTACACATATTCTCTGTC/5phos/ -5' After tagementation fragment options: single insertion not able to make a PCRable fragment Orange Blue 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Blue Orange 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Orange Orange 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Blue Blue 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' After fill in at 72 for 3 minutes (gap filling). Orange Blue 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3' 3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Blue Orange 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Orange Orange 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' 3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Blue Blue 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' Now PCRable, adding on adaptors. Adaptor 2 Blue Orange 3'- GACTCGCCCGACCGTTCCGTCTGGC[bc]TAGAGCATACGGCAGAAGACGAAC -5' 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' Adaptor 1 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG -3' Amplification with primer 1 and primer 2. Blue Orange (primer 2) 3'- AGCATACGGCAGAAGACGAAC -5' 5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[bc]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[bc]TAGAGCATACGGCAGAAGACGAAC -5' 5'- AATGATACGGCGACCACCGA -3' (primer 1) Nextera read primers for sequencing: Blue Orange 3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' (nextera read primer 2) 5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[bc]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[bc]TAGAGCATACGGCAGAAGACGAAC -5' (nextera read primer 1) 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' (nextera index read primer) 5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'