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== 3) Methylated adaptor ligation == * Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase ==== <u>Ligation reaction set up</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)<br> - Prepare ligation reaction mix (during drying the beads) ====<u> Ligation reaction Mix</u> ==== :{| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:70px;background:#f0f0f0;"|'''24rxn Mix''' |- | dA-tailed DNA||20.00||0.00 |- | 10x Ligation Buffer||3.00||72.00 |- | T4 DNA Ligase (30U/ul)||1.00||24.00 |- | TruSeq Adaptor DNA||1.00||24.00 |- | H2O||5.00||120.00 |- | Total||30.00|| |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Aliquot ligation reaction mix to 8-tube strip. 29ul for tube #1-3, #6-8 and 20ul for tube #4-5 :- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :{| {{table}} class = wikitable | align="center" style="width:90px;background:dodgerblue;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:90px;background:dodgerblue;;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' |- | 6-T-1.1||1||7-T-1.1||13 |- | 6-T-1.2||2||7-T-1.2||14 |- | 6-T-2.1||23||7-T-2.1||15 |- | 6-T-2.2||4||7-T-2.2||16 |- | 6-T-3.1||5||7-T-3.1||27 |- | 6-T-3.2||6||7-T-3.2||18 |- | 6-T-4.1||7||7-T-4.1||19 |- | 6-T-4.2||8||7-T-4.2||20 |- | 6-T-5.1||25||7-T-5.1||21 |- | 6-T-5.2||10||7-T-5.2||22 |- | 6-T-NTC||10||7-T-NTC||22 |} :- Add 9ul of ligation reaction mix with multichannel pipette :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 24h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C) :- Mix by gentle pulse-vortexing on PCR rack 5x :- Quickly spin down :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) * Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume diluted AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer<br> ==== 2014-06-29 ==== ==== <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 670ul of AMPure beads and 670ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT (forgot to dilute, used beads directly)<br> :- Add 60ul 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 31 EB Buffer. :- Wait for 2min. :- Transfer to sit on magnet for 2 min and transfer 30ul of adaptor-ligated DNA to new strip tubes for bisulfite conversion <u>(discard the beads)</u> :- Continue to bisulfite conversion <br>
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