Calendar/2012-7-13: Difference between revisions

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==qPCR with N2 Barcodes==
==qPCR with N2 Barcodes==
{| style="float: right;"
|- 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"
|style="text-decoration:none" height="15" | Template
| align="center" | 10
| Do not add to MM
|- style="font-size:8pt" align="center"
| height="15" | 5uM PCR F+R Barcode
| align="center" | 1
| Do not add to MM
|- style="font-size:8pt" align="center"
| height="15" | 50X SYBG
| align="center" | 0.2
| align="center" | 2.64
|- style="font-size:8pt" align="center"
| height="15" | 2X PhusionHF MM
| align="center" | 25
| align="center" | 330
|- style="font-size:8pt" align="center"
| height="15" | H20
| align="center" | 13.8
| align="center" | 182.16
|- style="font-size:8pt" align="center"
| height="15" | Total Volume
| align="center" | 50
| align="center" | 660
|}
{| 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="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | shTDG-IgG
|style="font-size:8pt" height="15" | shTDG-IgG
| align="center" | 8
|style="font-size:8pt" align="center" | 8
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | shTDG-5mC
|style="font-size:8pt" height="15" | shTDG-5mC
| align="center" | 9
|style="font-size:8pt" align="center" | 9
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | shTDG-5hmC
|style="font-size:8pt" height="15" | shTDG-5hmC
| align="center" | 10
|style="font-size:8pt" align="center" | 10
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | shTDG-5fC
|style="font-size:8pt" height="15" | shTDG-5fC
| align="center" | 11
|style="font-size:8pt" align="center" | 11
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|- style="font-size:8pt" align="center"
|- align="center"
| height="15" | shTDG-caC
|style="font-size:8pt" height="15" | shTDG-caC
| align="center" | 12
|style="font-size:8pt" align="center" | 12
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;
|style="font-size:12pt" align="center" | &nbsp;


|}
|}
*Add 20ul 10uM N2-bar-code primers into strip tube
*Add 20ul 10uM N2-bar-code primers into strip tube
*Add 20ul 10uM PCR_F primers and mix
*Add 20ul 10uM PCR_F primers and mix

Revision as of 01:41, 16 July 2012

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

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

Gel quant

Qubit

DIP Sample N2 Barcode Sample Concentration ng/ul Volume ul Amount ng
shCONT-input 1 3.62 30 108.6
shCONT-IgG 2 2.36 30 70.8
shCONT-5mC 3 2.64 30 79.2
shCONT-5hmC 4 3.54 30 106.2
shCONT-5fc 5 3.58 30 107.4
shCONT-5caC 6 3.82 30 114.6
shTDG-input 7 3.54 30 106.2
shTDG-IgG 8 2.4 30 72
shTDG-5mC 9 3.06 30 91.8
shTDG-5hmC 10 4.04 30 121.2
shTDG-5fC 11 3.9 30 117
shTDG-caC 12 3.18 30 95.4