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==Library Construction of Dr. Yi Zhang's DiP libraries Sample== ==Samples Preparation Protocol from Li== [[File:0712 DIP-seq samples.pdf]] *Received samples from Li on Jul 13 2012, 12 samples total in a yellow box, transfer to -20C ==qPCR with N2 Barcodes== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:8pt;font-weight:bold" align="center" |style="background-color:#FCD5B4;text-decoration:underline" width="90" height="15" | DIP Sample |style="background-color:#FCD5B4" width="65" | N2 Barcode |style="background-color:#B7DEE8" width="144" | Content |style="background-color:#B7DEE8" width="65" | Volume Per Reaction |style="background-color:#B7DEE8" width="65" | MM |- style="font-size:8pt" align="center" |style="text-decoration:none" height="15" | shCONT-input | align="center" | 1 | Template | align="center" | 10 | Do not add to MM |- style="font-size:8pt" align="center" | height="15" | shCONT-IgG | align="center" | 2 | 5uM PCR F+R Barcode | align="center" | 1 | Do not add to MM |- style="font-size:8pt" align="center" | height="15" | shCONT-5mC | align="center" | 3 | 50X SYBG | align="center" | 0.2 | align="center" | 2.64 |- style="font-size:8pt" align="center" | height="15" | shCONT-5hmC | align="center" | 4 | 2X PhusionHF MM | align="center" | 25 | align="center" | 330 |- style="font-size:8pt" align="center" | height="15" | shCONT-5fc | align="center" | 5 | H20 | align="center" | 13.8 | align="center" | 182.16 |- style="font-size:8pt" align="center" | height="15" | shCONT-5caC | align="center" | 6 | Total Volume | align="center" | 50 | align="center" | 660 |- align="center" |style="font-size:8pt" height="15" | shTDG-input |style="font-size:8pt" align="center" | 7 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |- align="center" |style="font-size:8pt" height="15" | shTDG-IgG |style="font-size:8pt" align="center" | 8 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |- align="center" |style="font-size:8pt" height="15" | shTDG-5mC |style="font-size:8pt" align="center" | 9 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |- align="center" |style="font-size:8pt" height="15" | shTDG-5hmC |style="font-size:8pt" align="center" | 10 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |- align="center" |style="font-size:8pt" height="15" | shTDG-5fC |style="font-size:8pt" align="center" | 11 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |- align="center" |style="font-size:8pt" height="15" | shTDG-caC |style="font-size:8pt" align="center" | 12 |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |style="background-color:#D9D9D9;font-size:12pt" align="center" | |} *Add 20ul 10uM N2-bar-code primers into strip tube *Add 20ul 10uM PCR_F primers and mix *We have 5uM F+R N2 Ind primer (Store at 4C Alice yellow 0.2ul tube rack) *Add 10ul template (for low volume sample add 5ul sample and add 5ul h2o) and 1ul barcode primers (using multi channel pipette) to strip tube *Add 39ul MM to each well 98C 30sec (98C 10sec -> 63C 20sec-> 72C 30sec) x 20 cycles 72C 2min 15C hold. ==qPCR Curves== <gallery widths=300px heights=300px perrow=7 caption="Samples"> File:120713_1.jpg|Samples taken out after cycle 7, A6-Ind1, D6 Ind4, G6 Ind7, D7, Ind 10 File:120713_2.jpg|C6 Ind3, C7 Ind9 File:120713_3.jpg|E6 Ind5, E7 Ind11 File:120713_4.jpg|F6 Ind6, F7 Ind12 File:120713_5.jpg|B6 Ind2, H6 Ind8 </gallery> ==Gel quant== <gallery widths=350px heights=350px perrow=7 caption="TBE Gel"> File:ZhangLab_2 2012-07-15 16hr 19min.jpg|LM space N2 Ind 1-9 File:ZhangLab_2 2012-07-15 16hr 20min.jpg|LM N2 Ind 10-12 </gallery> ==Qiaquick Purificaiton== *Elute with 30ul EB ==Qubit== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="background-color:#CCC0DA;font-size:8pt;font-weight:bold" align="center" |style="text-decoration:underline" width="90" height="15" | DIP Sample | width="65" | N2 Barcode | width="144" | Sample Concentration ng/ul | width="65" | Volume ul | width="65" | Amount ng | width="68" | To get 35 ng |- style="font-size:8pt" align="center" |style="text-decoration:none" height="15" | shCONT-input | align="center" | 1 | align="center" | 3.62 | align="center" | 30 | align="center" | 108.6 | align="center" | 9.67 |- style="font-size:8pt" align="center" | height="15" | shCONT-IgG | align="center" | 2 | align="center" | 2.36 | align="center" | 30 | align="center" | 70.8 | align="center" | 14.83 |- style="font-size:8pt" align="center" | height="15" | shCONT-5mC | align="center" | 3 | align="center" | 2.64 | align="center" | 30 | align="center" | 79.2 | align="center" | 13.26 |- style="font-size:8pt" align="center" | height="15" | shCONT-5hmC | align="center" | 4 | align="center" | 3.54 | align="center" | 30 | align="center" | 106.2 | align="center" | 9.89 |- style="font-size:8pt" align="center" | height="15" | shCONT-5fc | align="center" | 5 | align="center" | 3.58 | align="center" | 30 | align="center" | 107.4 | align="center" | 9.78 |- style="font-size:8pt" align="center" | height="15" | shCONT-5caC | align="center" | 6 | align="center" | 3.82 | align="center" | 30 | align="center" | 114.6 | align="center" | 9.16 |- style="background-color:#D8E4BC;font-size:8pt;font-weight:bold" align="center" |style="text-decoration:underline" height="15" | DIP Sample | N2 Barcode | Sample Concentration ng/ul | Volume ul | Amount ng | To get 35 ng |- style="font-size:8pt" align="center" |style="text-decoration:none" height="15" | shTDG-input | align="center" | 7 | align="center" | 3.54 | align="center" | 30 | align="center" | 106.2 | align="center" | 9.89 |- style="font-size:8pt" align="center" | height="15" | shTDG-IgG | align="center" | 8 | align="center" | 2.4 | align="center" | 30 | align="center" | 72 | align="center" | 14.58 |- style="font-size:8pt" align="center" | height="15" | shTDG-5mC | align="center" | 9 | align="center" | 3.06 | align="center" | 30 | align="center" | 91.8 | align="center" | 11.44 |- style="font-size:8pt" align="center" | height="15" | shTDG-5hmC | align="center" | 10 | align="center" | 4.04 | align="center" | 30 | align="center" | 121.2 | align="center" | 8.66 |- style="font-size:8pt" align="center" | height="15" | shTDG-5fC | align="center" | 11 | align="center" | 3.9 | align="center" | 30 | align="center" | 117 | align="center" | 8.97 |- style="font-size:8pt" align="center" | height="15" | shTDG-caC | align="center" | 12 | align="center" | 3.18 | align="center" | 30 | align="center" | 95.4 | align="center" | 11.01 |} *Pool Ind 1-6 in equal ratio *Pool Ind 7-12 in equal ratio ==Sample Tracking== *Original samples and Barcoded Samples transfered to -20C white box {{hly|yellow label "Yi Zhang Lab Dip samples Li Shen Jul 13 2012 Alan"}} *Pooled libraries transfered to sequencing sample box
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