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AlanFung:LabNotes/CTCF/2010-6-7
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==Prepare PCR Product from Jurkat gDNA== {| {{table}} | ||ul |- | primer||1 |- | h2o||7 |- | template||2 |- | Econo 2x||10 |- | |} *Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 2 -> 72C 10min -> 15C hold *Qiaquick Purification *Nanodrop P7-6.4ng/ul P10-5.4ng/ul *Gel Quant [[File:ZhangLab_2 2010-06-07 14hr 34min.jpg]] ===Protocol=== Obtain 100ng Jurkat DNA *100ng/(6.4ng/ul)=15.63ul *100ng/(5.4ng/ul)=18.52ul {| {{table}} | ||P7||P10 |- | Jurkat DNA PCR Product||15.63||18.52 |- | ddH2O||69.37||66.48 |- | End Repair Reaction Buffer (10x)||10||10 |- | Repair Enzyme Mix||5||5 |- | |} *Incubate 30 mins at 20C Purification using qiaquick column *Elute with 50ul buffer Perform A-tailing protocol (6/8/10) *Add in 6ul dA-tailing Reaction buffer *Add in 4ul klenow fragment *Incubate at 37C for 30mins Purification using qiaquick column *Elute with 50ul buffer Adapter ligation *Mix 1ul methylation adapter *Mix 5ul ligase *Mix 14ul ligase buffer *Incubate @20c for 30mins Purification using qiaquick column *Elute with 50ul buffer CT Conversion (6/3/10) *Take 46ul to perfrom CT conversion an additional 1m Bisulfite conversion of DNA *Add 46ul of sample to 130ul of CT conversion reagent solution in a pcr tube *Vortex the sample to mix *Pulse centrifuge *Perform *98C for 8m *64C for 3.5hr *4C hold *Add 600ul of M binding buffer into a column assembly *Load sample(s) to the column *Close the cap and mix by inverting the column several times *Centrifuge at >10,000g for 30sec *Discard the flow through *Add 100ul of M-Wash Buffer to the column *Centrifuge at full speed for 30sec *Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m *Centrifuge at full speed for 30sec *Add 100ul of M-Wash Buffer to the column *Centrifuge at full speed for 30sec *Place the column into a 1.5ml tube *Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements) *Centrifuge for 30sec to elute the DNA ==Perform PCR to bisulfite converted DNA using Phusion/iProof 6/9/10== *Primer {| {{table}} |- | PCR_F||Sol. Amp. (Forward)||AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] |- | PCR_R||Sol. Amp. (Reward)||CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] |- |} {| {{table}} | ||1rxn||4.4rxn|||| |- | Phusion||50||220|||| |- | PCRF||0.5||2.2|||| |- | PCRR||0.5||2.2|||| |- | SYBG||0.8||3.52|||| |- | |||||||| |- | ||P7||CTC P7||P10||CTC P10 |- | DNA||4||50||4||50 |- | H2O||46||0||46||0 |- | Mix||50||50||50||50 |- | |} a.Set up the reaction system with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 50ul 100uM PCR_F 0.5ul 100uM PCR_R 0.5ul 50X SYBG I 0.8ul Captured DNA 50ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. Terminate the reaction when the amplification curves approach to the plateau. [[File:060910.JPG|800px]] [[File:ZhangLab_2 2010-06-09 11hr 23min.jpg]] ==Conclusion== *Result indicates that library construction is successful using 100ng of input DNA, band ~308bp for P7 Primer and ~280bp for P10 primer (Expected size at + 33bpx2 to the pcr product size) *It also shows that after bisulfite conversion, libaries are lost during the process and could not be amplified *Indicating the we need to use carriers to protect libraries *Unspecific bands showing up ~130bp could be due to primer dimer issue *Is size selection needed? *Is it possible to use Kapa instead of Phusion? I haven't noticed non specific bands after amplification using KAPA ==Improvements== *Save a small amount of purified product after each step and run a gel to see which step brings in the non specific bands *Add carrier to protect libraries during bisulfite conversion *Size Selection *Using new Master Mix
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