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 |