Calendar/2012-7-13: Difference between revisions
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*Original samples and Barcoded Samples transfered to -20C white box {highlight:color=yellow}yellow label "Yi Zhang Lab Dip samples Li Shen Jul 13 2012 Alan"{highlight} | *Original samples and Barcoded Samples transfered to -20C white box {highlight:color=yellow}yellow label "Yi Zhang Lab Dip samples Li Shen Jul 13 2012 Alan"{highlight} | ||
*Pooled libraries transfered to sequencing sample box | *Pooled libraries transfered to sequencing sample box | ||
Revision as of 18:56, 16 July 2012
Library Construction of Dr. Yi Zhang's DiP libraries Sample
Samples Preparation Protocol from Li
- Received samples from Li on Jul 13 2012, 12 samples total in a yellow box, transfer to -20C
qPCR with N2 Barcodes
DIP Sample | N2 Barcode | Content | Volume Per Reaction | MM |
shCONT-input | 1 | Template | 10 | Do not add to MM |
shCONT-IgG | 2 | 5uM PCR F+R Barcode | 1 | Do not add to MM |
shCONT-5mC | 3 | 50X SYBG | 0.2 | 2.64 |
shCONT-5hmC | 4 | 2X PhusionHF MM | 25 | 330 |
shCONT-5fc | 5 | H20 | 13.8 | 182.16 |
shCONT-5caC | 6 | Total Volume | 50 | 660 |
shTDG-input | 7 | |||
shTDG-IgG | 8 | |||
shTDG-5mC | 9 | |||
shTDG-5hmC | 10 | |||
shTDG-5fC | 11 | |||
shTDG-caC | 12 |
- 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
- 120713 1.jpg
Samples taken out after cycle 7
- 120713 2.jpg
sample
- 120713 3.jpg
sample
- 120713 4.jpg
sample
- 120713 5.jpg
sample
Gel quant
- ZhangLab 2 2012-07-15 16hr 20min.jpg
LM N2 Ind 10-12
- ZhangLab 2 2012-07-15 16hr 19min.jpg
LM space N2 Ind 1-9
Qiaquick Purificaiton
- Elute with 30ul EB
Qubit
DIP Sample | N2 Barcode | Sample Concentration ng/ul | Volume ul | Amount ng | To get 35 ng |
shCONT-input | 1 | 3.62 | 30 | 108.6 | 9.67 |
shCONT-IgG | 2 | 2.36 | 30 | 70.8 | 14.83 |
shCONT-5mC | 3 | 2.64 | 30 | 79.2 | 13.26 |
shCONT-5hmC | 4 | 3.54 | 30 | 106.2 | 9.89 |
shCONT-5fc | 5 | 3.58 | 30 | 107.4 | 9.78 |
shCONT-5caC | 6 | 3.82 | 30 | 114.6 | 9.16 |
DIP Sample | N2 Barcode | Sample Concentration ng/ul | Volume ul | Amount ng | To get 35 ng |
shTDG-input | 7 | 3.54 | 30 | 106.2 | 9.89 |
shTDG-IgG | 8 | 2.4 | 30 | 72 | 14.58 |
shTDG-5mC | 9 | 3.06 | 30 | 91.8 | 11.44 |
shTDG-5hmC | 10 | 4.04 | 30 | 121.2 | 8.66 |
shTDG-5fC | 11 | 3.9 | 30 | 117 | 8.97 |
shTDG-caC | 12 | 3.18 | 30 | 95.4 | 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 {highlight:color=yellow}yellow label "Yi Zhang Lab Dip samples Li Shen Jul 13 2012 Alan"{highlight}
- Pooled libraries transfered to sequencing sample box