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== 2) End-repair/dA-tailing == * After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul * Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG * Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul :- Spin down the tubes :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tubes :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) :- Perform AMPure bead purification with 2x volume AMPure beads ==== <u>AMPure bead purification</u> ==== :- Add 60ul AMPure bead. Mix by pipetting 10x :- Wait for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH ::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 80% EtOH :- Dry the bead for 15min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u>
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