Rui:LabNotes/Collaborations/2011-10-11

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Revision as of 16:56, 13 October 2011 by >RuiLiu (→‎dA tailing)
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Illumina library preparation of samples for Dr. Xu Yang lab

DNA shearing

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

End repair

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

dA tailing

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

Adaptor ligation

  • 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

  • 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:
' ng/ul volume total
pre-7 2.86 20 57.2
post-7 10.6 15 159
pre-8 0.63 20 12.6
post-8 10.6 15 159
pre-9 6.18 20 123.6
post-9 8.47 15 127.05