Brandon:LabNotes/Project1/2012-5-7
Jump to navigation
Jump to search
Adaptation of transposition method for inserting T7
- in reference to shendure paper transposition
- also in reference to Athurva's summary
- new technique of only annealing sequences for form full mosaic ends and adding adaptors.
Summary and primers to order:
5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ spcr T7 forward spcr read sequence spcr ME 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ 3'-[ddC] CTACACATATTCTCTGTC [Phos]-5' 5'- [phos] CTGTCTCTTATACACATC [ddC]-3' 3’- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ Fragments created after transposition: 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNN------ CTGTCTCTTATACACATC -3' 3'- CTACACATATTCTCTGTC ------NNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ Second adapter to anneal: [phos] -5' CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNN------ CTGTCTCTTATACACATC -3' X X XXX X XXXX X XX XX XX XX X XXXX X XXX X X 3'- CTACACATATTCTCTGTC ------NNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ 3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC [phos] -5' Strands after fill-in, ligation, separation top strand 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ bottom strand 3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ read primers/amplification etc 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' read primer 1 3'- GACAGAGAATATGTGTAGAGACTACCTCGCCCGACCGT -5' read primer 2 AGCGGGCTGGCAAGGCAGACCGATC -3’
To order: 5’-[Phos]- CTGTCTCTTATACACATC -3’ [ddC - 3' to block ends]