Calendar/2012-7-13: Difference between revisions

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==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="background-color:#FCD5B4;font-size:8pt;font-weight:bold" align="center"
|- style="font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" width="76" height="15" | DIP Sample
|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"
| height="15" | shTDG-input
|style="font-size:8pt" height="15" | shTDG-input
| align="center" | 7
|style="font-size:8pt" align="center" | 7
 
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|- style="font-size:8pt" align="center"
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
| height="15" | shTDG-IgG
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
| align="center" | 8
 
|- style="font-size:8pt" align="center"
| height="15" | shTDG-5mC
| align="center" | 9
 
|- style="font-size:8pt" align="center"
| height="15" | shTDG-5hmC
| align="center" | 10
 
|- style="font-size:8pt" align="center"
| height="15" | shTDG-5fC
| align="center" | 11
 
|- style="font-size:8pt" align="center"
| height="15" | shTDG-caC
| align="center" | 12
 
|}
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#B7DEE8;font-size:8pt;font-weight:bold" align="center"
|style="text-decoration:underline" width="115" height="15" | Content
| width="110" | Volume Per Reaction
| width="98" | MM


|- style="font-size:8pt" align="center"
|- align="center"
|style="text-decoration:none" height="15" | Template
|style="font-size:8pt" height="15" | shTDG-IgG
| align="center" | 10
|style="font-size:8pt" align="center" | 8
| Do not add to MM
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
 
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|- style="font-size:8pt" align="center"
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
| height="15" | 5uM PCR F+R Barcode
| align="center" | 1
| Do not add to MM


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | 50X SYBG
|style="font-size:8pt" height="15" | shTDG-5mC
| align="center" | 0.2
|style="font-size:8pt" align="center" | 9
| align="center" | 2.64
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | 2X PhusionHF MM
|style="font-size:8pt" height="15" | shTDG-5hmC
| align="center" | 25
|style="font-size:8pt" align="center" | 10
| align="center" | 330
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | H20
|style="font-size:8pt" height="15" | shTDG-5fC
| align="center" | 13.8
|style="font-size:8pt" align="center" | 11
| align="center" | 182.16
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | Total Volume
|style="font-size:8pt" height="15" | shTDG-caC
| align="center" | 50
|style="font-size:8pt" align="center" | 12
| align="center" | 660
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;
|style="background-color:#D9D9D9;font-size:12pt" align="center" | &nbsp;


|}
|}
Line 106: Line 108:
   72C 2min   
   72C 2min   
   15C hold.
   15C hold.
==qPCR Curves==
==qPCR Curves==
[[File:120713_1.jpg|300x400px|thumb|sample]]
<gallery widths=300px heights=300px perrow=7 caption="Samples">
[[File:120713_2.jpg|300x400px|thumb|sample]]
File:120713_1.jpg|Samples taken out after cycle 7, A6-Ind1, D6 Ind4, G6 Ind7, D7, Ind 10
[[File:120713_3.jpg|300x400px|thumb|sample]]
File:120713_2.jpg|C6 Ind3, C7 Ind9
[[File:120713_4.jpg|300x400px|thumb|sample]]
File:120713_3.jpg|E6 Ind5, E7 Ind11
[[File:120713_5.jpg|300x400px|thumb|sample]]
File:120713_4.jpg|F6 Ind6, F7 Ind12
File:120713_5.jpg|B6 Ind2, H6 Ind8
</gallery>


==Gel quant==
==Gel quant==
*Ladder 10-12
<gallery widths=350px heights=350px perrow=7 caption="TBE Gel">
[[File:ZhangLab_2 2012-07-15 16hr 20min.jpg|400px]]
File:ZhangLab_2 2012-07-15 16hr 19min.jpg|LM space N2 Ind 1-9
*Ladder space N2 1-9
File:ZhangLab_2 2012-07-15 16hr 20min.jpg|LM N2 Ind 10-12
[[File:ZhangLab_2 2012-07-15 16hr 19min.jpg|400px]]
</gallery>
 
==Qiaquick Purificaiton==
*Elute with 30ul EB


==Qubit==
==Qubit==
Line 127: 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 134: 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 141: 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 148: 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 155: 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 162: 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 169: 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 183: 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 190: 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 197: 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 204: 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 211: 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]

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[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]

Gel quant[edit]

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