Revision as of 00:23, 11 July 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.
- Noi's experiment notes on wiki [[1]]
- Noi's wiki page shows that the library preparation failed and didn't have time to repeat experiment
- Dinh found the gDNA samples from Dr. Rana in Noi's box.
- From the labels, Noi got about 1 ug of the samples and only used 200 ng, we should have 800ng for library prep.
- 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, may be it is the expired beads used for purification but I will give a a try.
- I will repeat the library prep follow the exact protocol that you did with the tumor WGBS libraries. Using new beads and switch to Kapa Hyper for library preparation.
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)
|
Actual sample left
|
BR_1
|
21.7
|
18.43
|
91.57
|
Less than 10 ul
|
BR_2
|
54
|
7.41
|
102.59
|
OK
|
BR_3
|
57.3
|
6.98
|
103.02
|
OK
|
BR_4
|
71.9
|
5.56
|
104.44
|
OK
|
BR_5
|
39.5
|
10.13
|
99.87
|
Less than 10.13 but close
|
BR_6
|
29.4
|
13.61
|
96.39
|
Less than 10 ul
|
BR_7
|
33.7
|
11.87
|
98.13
|
Less than 10 ul
|
BR_8
|
38.3
|
10.44
|
99.56
|
Less than 10 ul
|
BR_9
|
32.1
|
12.46
|
97.54
|
Less than 10 ul
|
BR_10
|
24
|
16.67
|
93.33
|
Close to none remaining
|
BR_11
|
40.3
|
9.93
|
100.07
|
Less than 9.93 ul
|
BR_12
|
46.3
|
8.64
|
101.36
|
OK
|
BR_13
|
27.8
|
14.39
|
95.61
|
None Remaining
|
BR_14
|
29.5
|
13.56
|
96.44
|
Less than 10 ul
|
BR_15
|
35.2
|
11.36
|
98.64
|
Less than 10 ul
|
BR_16
|
39
|
10.26
|
99.74
|
Less than 10 ul
|
- 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 (2015-06-10)
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 (2015-06-10)
- 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.
Beads Purification (2015-06-10)
- 0.8X beads purification and elute with 25ul ddh2o.
- BR_X|Methylated|Adapter->Box(Alan 2015/6/3 Dr. Rana WGBS)
qPCR to check End Repair, A-tailing, and adapter ligation works (2015-06-11)
Components
|
1rxn
|
8.5 rxn mix
|
Adapter ligated DNA
|
2
|
Do not Add
|
2.5uM PCR_F+PCR_R N2 IndX
|
2
|
Do not Add
|
2x KAPA SYBG MM
|
12.5
|
233.75
|
H2O
|
8.5
|
158.95
|
Total
|
25
|
212.5
|
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold
File:AlanPCR12015611.jpg
- All samples are amplified except #13.
- BR_X|No Meth|PCR->Box(Alan 2015/6/3 Dr. Rana WGBS)
Bisulfite Conversion with Zymo Lighting Kit (2015-07-10)
- Add 130ul lighting conversion reagent to 20ul of sample, compensate with water if less than 20ul.
- Vortex and centrifuge.
- (98C 8min > 54C 60min > 10C hold)
- Add 600ul of M-Binding Buffer to column with collection tube.
- Load the sample to column. Close the cap and mix by inverting the column several times.
- Centrifuge at full speed for 30s. Discard flow through.
- Add 100 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30s.
- Add 200 μl of L-Desulphonation Buffer to the column and let stand at RT for 120 mins. After the incubation, centrifuge at full speed for 30 seconds.
- Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds.
- Repeat this wash step.
- Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
- The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.
PCR Amplification and Barcoding (2015-07-10)
Components
|
1rxn
|
17 rxn mix
|
Final Concentration
|
Adapter ligated Bisulfite Converted DNA
|
3
|
0
|
n/a
|
10uM PCR_F
|
0.5
|
9.35
|
200nM
|
10uM PCR_R.Ind2
|
0.5
|
9.35
|
200nM
|
2x KAPA SYBG MM
|
12.5
|
233.8
|
1X
|
H2O
|
8.5
|
159.0
|
n/a
|
Total
|
25
|
411.4
|
n/a
|
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold