Rui:LabNotes/Collaborations/2011-10-11
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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
|
TBE gel check[edit]
File:10.12.11 RNA.jpg