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== 2) End-repair/dA-tailing == '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 26ul of Klenow exo- with 26ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA<br> - Aliquot Klenow, exo- and dA:dC:dG mix to 8-tube strip. 7ul for tube #1-3, #6-8 and 5ul for tube #4-6 :- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. Pipette few times :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) * Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 1400ul of AMPure beads in 30mL reservoir and leave at RT<br> :- Add 64ul AMPure bead with multichannel pipette. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u><br> <br>
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