Calendar/2012-7-13: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
(39 intermediate revisions by the same user not shown) | |||
Line 7: | Line 7: | ||
==qPCR with N2 Barcodes== | ==qPCR with N2 Barcodes== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style=" | |- style="font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" width=" | |style="background-color:#FCD5B4;text-decoration:underline" width="90" height="15" | DIP Sample | ||
| width="65" | N2 Barcode | |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="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="15" | shCONT-input | |style="text-decoration:none" height="15" | shCONT-input | ||
| align="center" | 1 | | align="center" | 1 | ||
| Template | |||
| align="center" | 10 | |||
| Do not add to MM | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height="15" | shCONT-IgG | | height="15" | shCONT-IgG | ||
| align="center" | 2 | | align="center" | 2 | ||
| 5uM PCR F+R Barcode | |||
| align="center" | 1 | |||
| Do not add to MM | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height="15" | shCONT-5mC | | height="15" | shCONT-5mC | ||
| align="center" | 3 | | align="center" | 3 | ||
| 50X SYBG | |||
| align="center" | 0.2 | |||
| align="center" | 2.64 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height="15" | shCONT-5hmC | | height="15" | shCONT-5hmC | ||
| align="center" | 4 | | align="center" | 4 | ||
| 2X PhusionHF MM | |||
| align="center" | 25 | |||
| align="center" | 330 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height="15" | shCONT-5fc | | height="15" | shCONT-5fc | ||
| align="center" | 5 | | align="center" | 5 | ||
| H20 | |||
| align="center" | 13.8 | |||
| align="center" | 182.16 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
| height="15" | shCONT-5caC | | height="15" | shCONT-5caC | ||
| align="center" | 6 | | align="center" | 6 | ||
| Total Volume | |||
| align="center" | 50 | |||
| align="center" | 660 | |||
|- style="font-size:8pt" align="center" | |- align="center" | ||
| | |style="font-size:8pt" height="15" | shTDG-input | ||
| align="center" | | |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" | | |||
|- style="font-size:8pt" align="center" | |- align="center" | ||
| | |style="font-size:8pt" height="15" | shTDG-IgG | ||
| align="center" | | |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" | | |||
|- style="font-size:8pt" align="center" | |- align="center" | ||
| | |style="font-size:8pt" height="15" | shTDG-5mC | ||
| align="center" | | |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" | | |||
|- style="font-size:8pt" align="center" | |- align="center" | ||
| | |style="font-size:8pt" height="15" | shTDG-5hmC | ||
| align="center" | | |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" | | |||
|- style="font-size:8pt" align="center" | |- align="center" | ||
| | |style="font-size:8pt" height="15" | shTDG-5fC | ||
| align="center" | | |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" | | |||
| | |||
|style=" | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|} | |} | ||
Line 106: | Line 108: | ||
72C 2min | 72C 2min | ||
15C hold. | 15C hold. | ||
==qPCR Curves== | ==qPCR Curves== | ||
<gallery widths=300px heights=300px perrow=7 caption="Samples"> | <gallery widths=300px heights=300px perrow=7 caption="Samples"> | ||
File:120713_1.jpg | File:120713_1.jpg|Samples taken out after cycle 7, A6-Ind1, D6 Ind4, G6 Ind7, D7, Ind 10 | ||
File:120713_2.jpg| | File:120713_2.jpg|C6 Ind3, C7 Ind9 | ||
File:120713_3.jpg| | File:120713_3.jpg|E6 Ind5, E7 Ind11 | ||
File:120713_4.jpg| | File:120713_4.jpg|F6 Ind6, F7 Ind12 | ||
File:120713_5.jpg| | File:120713_5.jpg|B6 Ind2, H6 Ind8 | ||
</gallery> | </gallery> | ||
==Gel quant== | ==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== | ==Qubit== | ||
Line 129: | Line 135: | ||
| width="65" | Volume ul | | width="65" | Volume ul | ||
| width="65" | Amount ng | | width="65" | Amount ng | ||
| width="68" | To get 35 ng | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 136: | Line 143: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 108.6 | | align="center" | 108.6 | ||
| align="center" | 9.67 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 143: | Line 151: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 70.8 | | align="center" | 70.8 | ||
| align="center" | 14.83 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 150: | Line 159: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 79.2 | | align="center" | 79.2 | ||
| align="center" | 13.26 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 157: | Line 167: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 106.2 | | align="center" | 106.2 | ||
| align="center" | 9.89 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 164: | Line 175: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 107.4 | | align="center" | 107.4 | ||
| align="center" | 9.78 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 171: | Line 183: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 114.6 | | 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="font-size:8pt" align="center" | ||
| height="15" | shTDG-input | |style="text-decoration:none" height="15" | shTDG-input | ||
| align="center" | 7 | | align="center" | 7 | ||
| align="center" | 3.54 | | align="center" | 3.54 | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 106.2 | | align="center" | 106.2 | ||
| align="center" | 9.89 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 185: | Line 207: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 72 | | align="center" | 72 | ||
| align="center" | 14.58 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 192: | Line 215: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 91.8 | | align="center" | 91.8 | ||
| align="center" | 11.44 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 199: | Line 223: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 121.2 | | align="center" | 121.2 | ||
| align="center" | 8.66 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 206: | Line 231: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 117 | | align="center" | 117 | ||
| align="center" | 8.97 | |||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" | ||
Line 213: | Line 239: | ||
| align="center" | 30 | | align="center" | 30 | ||
| align="center" | 95.4 | | 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 |
Latest revision as of 19:41, 16 July 2012
Library Construction of Dr. Yi Zhang's DiP libraries Sample[edit]
Samples Preparation Protocol from Li[edit]
- Received samples from Li on Jul 13 2012, 12 samples total in a yellow box, transfer to -20C
qPCR with N2 Barcodes[edit]
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[edit]
- 120713 1.jpg
Samples taken out after cycle 7, A6-Ind1, D6 Ind4, G6 Ind7, D7, Ind 10
- 120713 2.jpg
C6 Ind3, C7 Ind9
- 120713 3.jpg
E6 Ind5, E7 Ind11
- 120713 4.jpg
F6 Ind6, F7 Ind12
- 120713 5.jpg
B6 Ind2, H6 Ind8
Gel quant[edit]
- ZhangLab 2 2012-07-15 16hr 19min.jpg
LM space N2 Ind 1-9
- ZhangLab 2 2012-07-15 16hr 20min.jpg
LM N2 Ind 10-12
Qiaquick Purificaiton[edit]
- Elute with 30ul EB
Qubit[edit]
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[edit]
- Original samples and Barcoded Samples transfered to -20C white box yellow label "Yi Zhang Lab Dip samples Li Shen Jul 13 2012 Alan"
- Pooled libraries transfered to sequencing sample box