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== Adaptor ligation == * Since I have different amount of DNA input, so I added different conc. of adaptor based on the range of input amount. * Dilute some adaptor to 1.5uM (10X) --> by mixing 3ul of adaptor with 27ul of 1X Stoffel Buffer :{| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Tube #''' | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock 15uM''' | align="center" style="width:80px;background:#f0f0f0;"|'''1.11X Stoffel Buffer''' |- | #1||Ind_3||3||27 |- | #2||Ind_7||3||27 |- | #3||Ind_8||3||27 |- | #4||Ind_9||3||27 |- | #5||Ind_13||3||27 |- | #6||Ind_14||3||27 |- | #7||Ind_15||3||27 |- | #8||Ind_16||3||27 |- | #9||Ind_20||3||27 |- | #10||Ind_22||3||27 |- | #11||Ind_23||3||27 |- | #12||Ind_25||3||27 |- | #13||Ind_27||3||27 |} * Actually H2O can be used to dilute adaptor {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''End repair/ dA-tailed DNA (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount (ng) used in WGBS/ KAPAHyper''' | align="center" style="width:80px;background:#f0f0f0;"|'''Adaptor stock (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:60px;background:#f0f0f0;"|'''Indx''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final Adaptor conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ligation rxn mix (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' |- | PCP_2||30||20||15000||1||PCP_2||4||1||273||20||55 |- | PCP_3||30||20||15000||1||PCP_3||4||2||273||20||55 |- | PCP_4||30||6.5||1500||5||PCP_4||0||3||136||20||55 |- | PCP_5||30||20||15000||1||PCP_5||4||4||273||20||55 |- | PCP_6||30||15||15000||1||PCP_6||4||5||273||20||55 |- | PCP_7||30||15||15000||1||PCP_7||4||6||273||20||55 |- | PCP_8||30||10||1500||5||PCP_8||0||7||136||20||55 |- | PCP_9||30||3.5||1500||2.5||PCP_9||2.5||8||68||20||55 |- | 6P-1||30||2.5||1500||2.5||6P-1||2.5||9||68||20||55 |- | 6P-2||30||20||15000||1||6P-2||4||10||273||20||55 |- | 6P-3||30||20||15000||1||6P-3||4||11||273||20||55 |- | 6P-4||30||20||15000||1||6P-4||4||12||273||20||55 |- | 6P-5||30||10||1500||5||6P-5||0||13||136||20||55 |- | 6P-8||30||3.2||1500||2.5||6P-8||2.5||14||68||20||55 |- | 6P-9||30||8.5||1500||5||6P-9||0||15||136||20||55 |- | 6P-10||30||6.4||1500||5||6P-10||0||16||136||20||55 |- | 7P-1||30||15||15000||1||7P-1||4||18||273||20||55 |- | 7P-2||30||20||15000||1||7P-2||4||19||273||20||55 |- | 7P-3||30||2.5||1500||2.5||7P-3||2.5||20||68||20||55 |- | 7P-5||30||20||15000||1||7P-5||4||21||273||20||55 |- | 7P-6||30||3.4||1500||2.5||7P-6||2.5||22||68||20||55 |- | 7P-7||30||3.3||1500||2.5||7P-7||2.5||23||68||20||55 |- | 7P-8||30||1.5||1500||2.5||7P-8||2.5||25||68||20||55 |- | 7-P-10||30||2.5||1500||2.5||7P-10||2.5||27||68||20||55 |- | NTC||30||NA||1500||5||NTC||0||27||136||20||55 |} ==== Adaptor ligation reaction mix ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''26x rxn''' |- | Ligation buffer||30.00||15.00||390.00 |- | DNA ligase||10.00||5.00||130.00 |- | Total||40.00||20.00|| |- | 1:20 diluted adaptor (714nM)||Varied||Varied|| |- | Total||110.00||55.00|| |}
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