Rui:LabNotes/Collaborations/2011-10-12
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Illumina library preparation of samples for Dr. Yi Zhang 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:
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
- 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 Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 10 cycles
- beads purification: 0.7X, elute in 15ul, take 12ul