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: | ||
Revision as of 16:57, 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: