Calendar/2012-7-13

From ZhangLabWiki
Revision as of 01:11, 16 July 2012 by >Alan6017518 (→‎qPCR Curves)
Jump to navigation Jump to search

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
shCONT-input 1
shCONT-IgG 2
shCONT-5mC 3
shCONT-5hmC 4
shCONT-5fc 5
shCONT-5caC 6
shTDG-input 7
shTDG-IgG 8
shTDG-5mC 9
shTDG-5hmC 10
shTDG-5fC 11
shTDG-caC 12
Content Volume Per Reaction MM
Template 10 Do not add to MM
5uM PCR F+R Barcode 1 Do not add to MM
50X SYBG 0.2 2.64
2X PhusionHF MM 25 330
H20 13.8 182.16
Total Volume 50 660
  • 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

File:120713 1.jpg
Samples taken out after cycle 7

Gel quant

  • Ladder 10-12

File:ZhangLab 2 2012-07-15 16hr 20min.jpg

  • Ladder space N2 1-9

File:ZhangLab 2 2012-07-15 16hr 19min.jpg

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