Rui:LabNotes/Collaborations/2011-10-11

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Illumina library preparation of samples for Dr. Xu Yang lab[edit]

DNA shearing[edit]

  • Follow the protocol used on 7/8/11 [1]
  • Morries Cancer Center, lab bay 2R (Yang or Sophie to contact)

End repair[edit]

  • Follow the protocol used on 7/11/11 [2]
  • Elute in 32ul and 1ul for Qubit measurement:

dA tailing[edit]

  • Follow the protocol used on 7/11/11 [3]
  • Elute in 20ul and 1ul for Qubit measurement:

Adaptor ligation[edit]

  • Follow the protocol used on 7/11/11 [4]
  • 20uM fresh made adaptor, ~1:20 ratio
  • Calculation for adaptor input:
' ng/ul volume total ng pmole adaptor ul add
#7 3.91 18 70.38 0.5414 10.83 0.54 0.5
#8 1.76 18 31.68 0.2437 4.87 0.24 0.5
#9 8.89 18 160.02 1.2309 24.62 1.23 1.2

PCR amplification[edit]

  • After ligation and beads purification, 25ul EB is used to elute DNA, 23ul to PCR
  • 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 8 cycles
  • Pre- and post-PCR measurement:

Concentration monitor[edit]

' Sample #7 #8 #9
pre-shearing ng/ul
volume
total ng
after repair ng/ul 3.03 0.665 6.63
volume 30 30 30
total ng 90.9 19.95 198.9
after A tailing ng/ul 3.91 1.76 8.89
volume 18 18 18
total ng 70.38 31.68 160.02
after ligation ng/ul 2.86 0.63 6.18
volume 20 20 20
total ng 57.2 12.6 123.6
after PCR ng/ul 10.6 10.6 8.47
volume 15 15 15
total ng 159 159 127.05
fold change 2.77972028 12.61904762 1.027912621
pre-PCR ng 12
post PCR ng/ul 10
volume 15
total ng 150
fold change 12.5

TBE gel check[edit]

File:10.12.11 RNA.jpg