Annealing Oligos

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This is the procedure that we tried for annealing oligos that had 12 bp inverted repeats:

Step 1: Resuspend oligos @ 0.25 nmol/ul (eg. 52 nmol in 204 ul dH2O)

Step 2: Prepare 10x annealing buffer (1M NaCl; 100mM Tris-HCl, pH 7.4)

Step 3: 20 ul top strand, 20 ul bottom strand, 5 ul H2O, 5 ul 10x annealing buffer

Step 4: Thermal cycler --> 35 cycles of 95 C for 2 minutes, step down 2 C each cycle (In 70 minutes temp. goes from 95 C to 25 C)

-We used 1 ul(~4 ng) in ligation to 50 ng linearized vector. -Store @ -20 C

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