Rui:LabNotes/Collaborations/2011-10-11
Jump to navigation
Jump to search
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 |