Rui:LabNotes/Collaborations/2011-10-11: Difference between revisions

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* 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 8 cycles
* 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:
* Pre- and post-PCR measurement:
===Concentration monitor===
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''#7'''
| align="center" style="background:#f0f0f0;"|'''#8'''
| align="center" style="background:#f0f0f0;"|'''#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
|}

Revision as of 17:43, 13 October 2011

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:

Concentration monitor

' 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