Calendar/2012-7-13: Difference between revisions

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>Alan6017518
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Revision as of 16:47, 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 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
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