Revision as of 18:14, 17 September 2014 by >Noi
Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair)[edit]
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Zhang lab ID
|
Conc. (ng/ul)
|
Volume for 1ng
|
H2O
|
# in strip
|
Exp ID
|
Klenow,exo-:dAdCdGdT Mix
|
Total
|
NC-1 |
0.293 |
3.41 |
6.59 |
#1_1 |
wNC-1 |
10.00 |
20.00
|
NC-2 |
0.325 |
3.08 |
6.92 |
#1_2 |
wNC-2 |
10.00 |
20.00
|
NC-3 |
0.538 |
1.86 |
8.14 |
#1_3 |
wNC-3 |
10.00 |
20.00
|
NC-5 |
0.156 |
6.41 |
3.59 |
#1_4 |
wNC-5 |
10.00 |
20.00
|
NC-6 |
0.148 |
6.76 |
3.24 |
#1_5 |
wNC-6 |
10.00 |
20.00
|
NC-7 |
0.245 |
4.08 |
5.92 |
#1_6 |
wNC-7 |
10.00 |
20.00
|
NC-8 |
0.141 |
7.09 |
2.91 |
#1_7 |
wNC-8 |
10.00 |
20.00
|
NC-9 |
0.171 |
5.85 |
4.15 |
#1_8 |
wNC-9 |
10.00 |
20.00
|
NC-12 |
0.163 |
6.13 |
3.87 |
#2_1 |
wNC-12 |
10.00 |
20.00
|
NC-13 |
0.168 |
5.95 |
4.05 |
#2_2 |
wNC-13 |
10.00 |
20.00
|
NC-14 |
0.157 |
6.37 |
3.63 |
#2_3 |
wNC-14 |
10.00 |
20.00
|
NC-15 |
0.118 |
8.47 |
1.53 |
#2_4 |
wNC-15 |
10.00 |
20.00
|
NC-16 |
0.140 |
7.14 |
2.86 |
#2_5 |
wNC-16 |
10.00 |
20.00
|
NC-17 |
0.126 |
7.94 |
2.06 |
#2_6 |
wNC-17 |
10.00 |
20.00
|
NC-18 |
0.189 |
5.29 |
4.71 |
#2_7 |
wNC-18 |
10.00 |
20.00
|
NC-19 |
0.105 |
9.52 |
0.48 |
#2_8 |
wNC-19 |
10.00 |
20.00
|
NC-20 |
0.128 |
7.81 |
2.19 |
#3_1 |
wNC-20 |
10.00 |
20.00
|
NC-21 |
0.205 |
4.88 |
5.12 |
#3_2 |
wNC-21 |
10.00 |
20.00
|
NC-22 |
0.180 |
5.56 |
4.44 |
#3_3 |
wNC-22 |
10.00 |
20.00
|
NC-23 |
0.153 |
6.54 |
3.46 |
#3_4 |
wNC-23 |
10.00 |
20.00
|
NC-24 |
0.140 |
7.14 |
2.86 |
#3_5 |
wNC-24 |
10.00 |
20.00
|
NC-27 |
0.124 |
8.06 |
1.94 |
#3_6 |
wNC-27 |
10.00 |
20.00
|
NC-29 |
0.109 |
9.17 |
0.83 |
#3_7 |
wNC-29 |
10.00 |
20.00
|
NC-30 |
0.099 |
10.00 |
0.00 |
#3_8 |
wNC-30 |
10.00 |
20.00
|
NTC_1 |
0 |
0.00 |
10.00 |
#4_1 |
|
10.00 |
20.00
|
NTC_2 |
0 |
0.00 |
10.00 |
#4_2 |
|
10.00 |
20.00
|
NC-3 |
0.538 |
1.86 |
8.14 |
#4_3 |
wNC-3t |
10.00 |
20.00
|
NC-4 |
0.848 |
1.18 |
8.82 |
#4_4 |
wNC-4t |
10.00 |
20.00
|
- The last two samples, NC-3 and NC-4 were used to tested for T4 DNA ligase HC that has been delivered during weekend (Saturday) and was left at RT until Monday. I got the new tube from Thermoscientific for replacement. All samples except the last two were prepared with the new tube of T4 DNA ligase HC.
- Prepare new tube of dA:dC:dG:dT (20:2:2:2 mM)
Components
|
Final conc. (nM)
|
Volume (ul)
|
100mM dATP |
20 |
100
|
100mM dCTP |
2 |
10
|
100mM dGTC |
2 |
10
|
100mM dTTC |
2 |
10
|
H2O |
|
370
|
Klenow exo- and dA:dG:dC reaction mix[edit]
Components
|
1x rxn
|
30x rxn
|
10X Tango buffer |
2.00 |
60.00
|
dA:dC:dG:dT (20:2:2:2mM) |
1.00 |
30.00
|
Klenow fragment, exo- (5U/ul) |
1.00 |
30.00
|
H2O |
6.00 |
180.00
|
Total |
10.00 |
|
- Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT reaction mix to 8-tube strip
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification
Ligation reaction mix[edit]
Exp ID
|
TruSeq Index
|
Exp ID
|
TruSeq Index
|
Exp ID
|
TruSeq Index
|
Exp ID
|
TruSeq Index
|
wNC-1 |
1 |
wNC-12 |
13 |
wNC-20 |
11 |
NTC_1 |
3
|
wNC-2 |
2 |
wNC-13 |
14 |
wNC-21 |
12 |
NTC_2 |
9
|
wNC-3 |
4 |
wNC-14 |
15 |
wNC-22 |
21 |
wNC-3t |
23
|
wNC-5 |
5 |
wNC-15 |
16 |
wNC-23 |
22 |
wNC-4t |
25
|
wNC-6 |
6 |
wNC-16 |
27 |
wNC-24 |
3 |
|
|
wNC-7 |
7 |
wNC-17 |
18 |
wNC-27 |
9 |
|
|
wNC-8 |
8 |
wNC-18 |
19 |
wNC-29 |
23 |
|
|
wNC-9 |
10 |
wNC-19 |
20 |
wNC-30 |
25 |
|
|
Ligation reaction mix[edit]
Components
|
1x rxn
|
28x rxn
|
dA-tailed DNA |
20.00 |
0.00
|
10X Tango buffer |
0.50 |
14.00
|
HC T4 DNA ligase (30units/ul) |
1.00 |
28.00
|
10mM ATP |
1.25 |
35.00
|
H2O |
1.25 |
35.00
|
Total |
24.00 |
|
Components
|
1x rxn
|
2x rxn
|
dA-tailed DNA |
20.00 |
0.00
|
10X Tango buffer |
0.50 |
1.00
|
THAWED HC T4 DNA ligase (30units/ul) |
1.00 |
2.00
|
10mM ATP |
1.25 |
2.50
|
H2O |
1.25 |
2.50
|
Total |
24.00 |
|
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 4ul of ligation reaction mix with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion
Bisulfite conversion[edit]
Bisulfite conversion procedures
Amplification[edit]
- Repeat all procedure exactly the same as previous experiment on 2014-08-03
1st round PCR (fix 12 cycles)[edit]
Components
|
Conc
|
unit
|
Final conc./amount
|
unit
|
Volume (ul)
|
Bis-cvt DNA |
|
|
|
|
30.00
|
10X Reaction buffer |
10 |
X |
1 |
X |
5.00
|
dNTP mix |
10 |
mM |
0.25 |
mM |
1.25
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
1.50
|
50X SYBG |
50 |
X |
0.4 |
X |
0.40
|
PfuTurbo Cx |
2.5 |
Unit/ul |
1 |
unit |
1.00
|
H2O |
|
|
|
|
10.85
|
Total |
|
|
|
|
50.00
|
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min
- - Purify with 1.25X AMPure beads
- - Elute with 42ul EB Buffer
- - Continue to 2nd round PCR. No PAGE verification.
2nd round PCR[edit]
Quick test[edit]
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
30 rxn mix
|
Purified 1st round DNA |
|
|
|
|
3.00 |
0.00
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
2.00 |
60.00
|
dNTP mix |
10 |
mM |
0.25 |
mM |
0.25 |
7.50
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
0.30 |
9.00
|
50X SYBR |
50 |
X |
0.4 |
X |
0.08 |
2.40
|
Phusion HF |
2 |
unit/ul |
|
|
0.10 |
3.00
|
H2O |
|
|
|
|
4.27 |
128.10
|
Total |
|
|
|
|
10.00 |
|
- - Aliquot 7 + 3ul DNA template
- - Mix well.
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- - Added 2ul 6X loading dye to each strip tube & mix well
- - Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel1.jpg File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel2.jpg
File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel3.jpg
- From quick qPCR test, there are variation of the intensity of libraries size ~300bp. 8 cycles seem to be over-amplified for almost samples. There are some samples that had very faint intensity of the libraries. I need to varied PCR cycle number for some samples.
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
27 rxn mix
|
Purified 1st round DNA |
|
|
|
|
36.00 |
0.00
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
24.00 |
648.00
|
dNTP mix |
10 |
mM |
0.25 |
mM |
3.00 |
81.00
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
3.60 |
97.20
|
50X SYBR |
50 |
X |
0.4 |
X |
0.96 |
25.92
|
Phusion HF |
2 |
unit/ul |
|
|
1.20 |
32.40
|
H2O |
|
|
|
|
51.24 |
1383.48
|
Total |
|
|
|
|
120.00 |
|
- - Aliquot 84 + 36ul DNA template
- - Mix well. Split 38ul X3
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 cycles --> 72C for 2min
- - I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer.
- - Loaded 2ul in 6% TBE gel for PAGE verification
File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel1.jpg File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel2.jpg
File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel3.jpg
- Different from WGBS from cancer patient small fragment DNA, there are high variations of library amount among different samples as mentioned from quick qPCR. Also, the last two samples (NC-3 and NC-4) ligated with T4 DNA ligase left at RT almost 2 days had comparable result compare to the same sample ligated with proper stored T4 DNA ligase.
Qubit dsDNA HS quantification of WGBS libraries from normal control cfDNA[edit]
Sample
|
Conc. in the Qubit
|
Unit
|
uL used
|
Dilution
|
Conc. (ng/ul)
|
Volume (ul)
|
Total amount (ng)
|
Used for prev. exp (ng)
|
Amount left (ng)
|
> 400ng
|
NC-1 |
4.1 |
ng/mL |
2 |
100 |
0.41 |
80.00 |
32.80 |
0.00 |
32.80 |
NO
|
NC-2 |
13.9 |
ng/mL |
2 |
100 |
1.39 |
80.00 |
111.20 |
0.00 |
111.20 |
NO
|
NC-3 |
18 |
ng/mL |
2 |
100 |
1.80 |
80.00 |
144.00 |
0.00 |
144.00 |
NO
|
NC-5 |
4.71 |
ng/mL |
2 |
100 |
0.47 |
80.00 |
37.68 |
0.00 |
37.68 |
NO
|
NC-6 |
42.5 |
ng/mL |
2 |
100 |
4.25 |
80.00 |
340.00 |
100.00 |
240.00 |
NO
|
NC-7 |
4.69 |
ng/mL |
2 |
100 |
0.47 |
80.00 |
37.52 |
0.00 |
37.52 |
NO
|
NC-8 |
15.2 |
ng/mL |
2 |
100 |
1.52 |
80.00 |
121.60 |
0.00 |
121.60 |
NO
|
NC-9 |
37.2 |
ng/mL |
2 |
100 |
3.72 |
80.00 |
297.60 |
100.00 |
197.60 |
NO
|
NC-12 |
29.9 |
ng/mL |
2 |
100 |
2.99 |
80.00 |
239.20 |
0.00 |
239.20 |
NO
|
NC-13 |
16.4 |
ng/mL |
2 |
100 |
1.64 |
80.00 |
131.20 |
0.00 |
131.20 |
NO
|
NC-14 |
12.8 |
ng/mL |
2 |
100 |
1.28 |
80.00 |
102.40 |
0.00 |
102.40 |
NO
|
NC-15 |
12.9 |
ng/mL |
2 |
100 |
1.29 |
80.00 |
103.20 |
0.00 |
103.20 |
NO
|
NC-16 |
13.3 |
ng/mL |
2 |
100 |
1.33 |
80.00 |
106.40 |
0.00 |
106.40 |
NO
|
NC-17 |
38.6 |
ng/mL |
2 |
100 |
3.86 |
80.00 |
308.80 |
0.00 |
308.80 |
NO
|
NC-18 |
26.3 |
ng/mL |
2 |
100 |
2.63 |
80.00 |
210.40 |
0.00 |
210.40 |
NO
|
NC-19 |
4.74 |
ng/mL |
2 |
100 |
0.47 |
80.00 |
37.92 |
0.00 |
37.92 |
NO
|
NC-20 |
3.96 |
ng/mL |
2 |
100 |
0.40 |
80.00 |
31.68 |
0.00 |
31.68 |
NO
|
NC-21 |
4.13 |
ng/mL |
2 |
100 |
0.41 |
80.00 |
33.04 |
0.00 |
33.04 |
NO
|
NC-22 |
27.1 |
ng/mL |
2 |
100 |
2.71 |
80.00 |
216.80 |
0.00 |
216.80 |
NO
|
NC-23 |
17.6 |
ng/mL |
2 |
100 |
1.76 |
80.00 |
140.80 |
0.00 |
140.80 |
NO
|
NC-24 |
3.87 |
ng/mL |
2 |
100 |
0.39 |
80.00 |
30.96 |
0.00 |
30.96 |
NO
|
NC-27 |
68.8 |
ng/mL |
2 |
100 |
6.88 |
80.00 |
550.40 |
100.00 |
450.40 |
YES
|
NC-29 |
35.9 |
ng/mL |
2 |
100 |
3.59 |
80.00 |
287.20 |
0.00 |
287.20 |
NO
|
NC-30 |
64.6 |
ng/mL |
2 |
100 |
6.46 |
80.00 |
516.80 |
100.00 |
416.80 |
YES
|