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==Adaptor ligation== Prepare 30uM adaptors: 100uM PE-t: 30ul 100uM PE-b: 30ul 10x stoffel buffer: 10ul H2O: 30ul 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec. commonly used adaptors: Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors (for the one adaptor protocol): 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s regular Y adaptor: PE_t_adaptor(top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond PE_b_adaptor(bottom) \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. ordering high quality HPLC purified adaptor will help reduce self-ligated adaptor sequences, thus eliminate the 125bp chimeric band 5x Kapa Ligation buffer 10ul DNA ligase 5ul DNA adaptor (30uM) 5ul A tailed DNA 30ul Incubate at 20C for 20 minutes purify the product with Ampure beads (1X) and elute in 42ul ddH2O
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