Rui:LabNotes/Collaborations/2011-10-12

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Illumina library preparation of samples for Dr. Yi Zhang 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:
samples ng/ul volume total ng pmole adaptor ul adjusted ul
1 3.28 18 59.04 0.4542 9.0831 0.4542 0.8
2 3.55 18 63.9 0.4915 9.8308 0.4915 0.8
3 4.75 18 85.5 0.6577 13.1538 0.6577 1
4 5.16 18 92.88 0.7145 14.2892 0.7145 1
5 2.44 18 43.92 0.3378 6.7569 0.3378 0.5
6 0.39 18 6.93 0.0533 1.0662 0.0533 0.5

PCR amplification[edit]

  • After ligation and beads purification, 22ul EB is used to elute DNA, take 20ul into tubes, take 5ul of s1-5 for PCR, use all (20ul) of s6
  • 50ul PCR reaction: 25ul Phusion, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 10 cycles
  • beads purification: 0.7X, elute in 15ul, take 12ul

Concentration monitor[edit]

' Sample #1 #2 #3 #4 #5 #6
pre-shearing ng/ul 16.7 23.6 22 11.2 1.55
volume 10 10 10 10 10 10
total ng 167 236 220 112 15.5
after repair ng/ul 2.78 2.7 3.63 3.98 2.16 0.3
volume 30 30 30 30 30 30
total ng 83.4 81 108.9 119.4 64.8 9.12
after A tailing ng/ul 3.28 3.55 4.75 5.16 2.44 0.39
volume 18 18 18 18 18 18
total ng 59.04 63.9 85.5 92.88 43.92 6.93
after ligation ng/ul 2.47 2.66 3.13 4 1.83 0.32
volume 20 20 20 20 20 20
total ng 49.4 53.2 62.6 80 36.6 6.32
pre-PCR ng 12.35 13.3 15.65 20 9.15 6.32
after PCR ng/ul 8.51 10.9 10.1 11.8 11.9 11.9
volume 15 15 15 15 15 15
total ng 127.65 163.5 151.5 177 178.5 178.5
fold change 10.33603239 12.29323308 9.680511182 8.85 19.50819672 28.24367089