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AlanFung:LabNotes/CTCF/2010-4-23
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==Project Update== ===Bottleneck=== *Extreme low amount of ligated product *Low adapter ligation efficiency ===Improvements=== *Start of with high amount of input DNA (500ng & 1ug,50ng) *Increase the amount of adapter and increase annealing time ==Quantification of Jurkat PCR Product== Qubit: 48.4ng/ul ==Library Prep with new Jurkat PCR product== *Concentration:48.4ng/ul *BP:100BP *setup: 1ug, 500ng and -ve control ==End Repair== {| {{table}} | ||1ug||500ng||50ng||-ve |- | DNA||20.66||10.33||1.03||0 |- | End Repair Buffer||10||10||10||10 |- | End Repair Enzyme Mix||5||5||5||5 |- | ddh2o||64.34||74.67||83.97||85 |- | Total Volume||100||100||100||100 |- | |} *Incubate at 20C for 30 minutes *EtOH purification resolve with 42ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue) ==A-Tailing== {| {{table}} | ||1ug||500ng||50ng||-ve |- | End Repaired DNA||42||42||42||42 |- | A-tailing Buffer||5||5||5||5 |- | Klenow Fragment||3||3||3||3 |- | Total Volume||50||50||50||50 |- | |} *Incubate at 37C for 30mins *EtOH purification resolve with 30ul ddh2o(125ul 100%EtOH, 5ul 3M NaOH, 1ul Glycoblue) ==Adapter Ligation== {| {{table}} | ||1ug||500ng||50ng||-ve |- | A-tailed DNA||30||30||30||30 |- | Ligation Buffer||10||10||10||10 |- | Methylation Adapter||5||5||5||5 |- | DNA Ligase||5||5||5||5 |- | Total Volume||50||50||50||50 |- | |} *Incubate at 20C for 30mins *EtOH purification resolve with 30ul ddh2o ==TBU Gel Quantification== *2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer *0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer *Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min *Pre run gel [[File:ZhangLab_2 2010-04-26 15hr 14min.jpg|500px]] ==Bead Purification== *Resolve with 40ul ==TBU Gel Quantification== *2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer *0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer *Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min *Pre run gel [[File:ZhangLab_2 2010-04-26 17hr 47min.jpg|500px]] *Quantify result for 500ng bands *2.3ng-middle band *2.12ng-upperband *The amount of DNA loaded to the gel may have overloaded the gel resulting in the big smear in the image *amount of ligated product = 40ng
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