Revision as of 00:36, 11 June 2015 by >Alan6017518
Background
- Noi made a batch of WGBS libraries on the whole blood DNA from Dr. Rana’s lab.
- Dr. Rana has obtained some sequencing fund, and would like to get them sequenced.
- If the libraries are good, can you tell Alan where to locate those libraries, so that we can send them to BioGem?
- Noi's wiki [[1]]
- Dinh found the gDNA samples from Dr. Rana in Noi's box.
- Noi's wiki page shows that the library preparation failed and didn't have time to repeat experiment [[2]]
- From the labels, Noi got about 1 ug of the samples and only used 200 ng, we should have 800ng for library prep.
- I will repeat the library prep follow the exact protocol that you did with the tumor WGBS libraries?
- Noi confirmed that the experiment failed for the first round of experiment and she did try a second time and still the result was not very good
- She got very faint smear or barely see a smear in many samples by amplifying about 16-18 cycles, which is very unlikely for DNA input ~100ng
- Noi doesn't know why this is the case but I will give a a try.
Covaris gDNA shearing (2015-06-09)
- Called IGM to reserve the Covaris Shearing Machine.
- I will shear 400ng of samples in case we need to repeat experiment again.
- Transfer 400ng of samples and make up to 110ul with ddh2o
Sample
|
Sample conc. (ng/ul)
|
Volume for 400ng (ul)
|
H2O (ul)
|
BR_1
|
21.7
|
18.43
|
91.57
|
BR_2
|
54
|
7.41
|
102.59
|
BR_3
|
57.3
|
6.98
|
103.02
|
BR_4
|
71.9
|
5.56
|
104.44
|
BR_5
|
39.5
|
10.13
|
99.87
|
BR_6
|
29.4
|
13.61
|
96.39
|
BR_7
|
33.7
|
11.87
|
98.13
|
BR_8
|
38.3
|
10.44
|
99.56
|
BR_9
|
32.1
|
12.46
|
97.54
|
BR_10
|
24
|
16.67
|
93.33
|
BR_11
|
40.3
|
9.93
|
100.07
|
BR_12
|
46.3
|
8.64
|
101.36
|
BR_13
|
27.8
|
14.39
|
95.61
|
BR_14
|
29.5
|
13.56
|
96.44
|
BR_15
|
35.2
|
11.36
|
98.64
|
BR_16
|
39
|
10.26
|
99.74
|
- Transfer to Covaris tube at IGM
- Perform shearing
Followed up the set up for Covaris shearing from previous experiment
Target BP 400
Tube Micro tube
Duty Factor 10%
Peak Incident Power (w) 140
Cycle per Burst 200
Time (secs) 55
Volume 50ul
Temperature (c) 7
Intensifier Yes
- Transfer sheared samples to non-stick 1.5mL tubes and label.BR_X|Sheared|400bp->Box(Alan 2015/6/3 Dr. Rana WGBS)
End Repairing and A-Tailing
Component
|
Volume (uL)
|
Master Mix (uL)
|
Fragment avg 400bp, double-stranded DNA
|
50
|
Do not add
|
End Repair & A-Tailing Buffer
|
7
|
130.9
|
End Repair & A-Tailing Enzyme Mix
|
3
|
56.1
|
Total Volume
|
60
|
187
|
- Premix buffer and enzyme mastermix in a tube, aliquot 10ul to each well in a strip tube.
- Add 50ul samples to each well and mix by pipetting.
Step
|
Temp
|
Time
|
End Repair
|
20 °C
|
30 min
|
A-Tailing
|
65 °C
|
30 min
|
HOLD
|
12 °C
|
∞
|
- Proceed immediately to the next step
Adapter Ligation
- Assemble each Adapter Ligation reaction as follows:
Component
|
Volume (μl)
|
MasterMix (μl)
|
End Repair & A-Tailing reaction product
|
60
|
Do not add
|
PCR-grade water
|
9
|
168.3
|
Ligation Buffer
|
30
|
561
|
DNA Ligase
|
10
|
187
|
Adapter stock
|
1
|
18.7
|
Total volume
|
110
|
935
|
- Incubate at 20 °C for 15 min.
- Proceed immediately to the next step.