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==Transposon annealing== *usually do it in EB buffer, Trina recommended to have 50 mM NaCl in it. *resuspend oligos in TE buffer. *annealing buffer (final concentration in annealing reaction): 50 mM NaCl 1X TE '''original protocol''' 1. annealing of ME sequence to T7 transposon sequence a. Make 100 uM stock solution of top oligo and bottom oligo. b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL. c. cool to RT at 0.1 C/s '''with NaCl''' annealing buffer (1.25X) 1.56 uL 1M NaCl (for 62.5 uM) 5 uL 5X TE (for 1.25X) 13.44 uL Nuclease free water 1. annealing of ME sequence to T7 transposon sequence a. Make 100 uM stock solution of top oligo and bottom oligo. b. Incubate 2.5 uL of each oligo (100uM) with 20 uL annealing buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL. c. cool to RT at 0.1 C/s
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