Revision as of 20:19, 8 June 2014 by >Noi
MONOD V1N3 BSPP capture (repeat sensitivity test)[edit]
Link to calendar: [[1]]
- I repeated MONOD V1N3 capture experiment on 3014-05-29: [[2]]
- I reduced capture volume to 10ul.
- I reduced incubation time after adding SLN mix between 4-6h since I think that 20h is not necessary for polymerization and may cause more back ground if DNA template level is too low.
- I will add experimental procedures later
Bisulfite conversion[edit]
Mix
|
82.1ng/ul JK DNA
|
H2O
|
Aliquot
|
25ng |
1.28 |
82.72 |
20.00
|
50ng |
2.56 |
81.44 |
20.00
|
100ng |
5.12 |
78.88 |
20.00
|
200ng |
10.23 |
73.77 |
20.00
|
Sample
|
Conc. (ng/ul)
|
Amount (ng)
|
Volume (ul)
|
H2O (ul)
|
Lightnig Conversion reagent
|
Total
|
JK-1 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-2 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-3 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-4 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-5 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-6 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-7 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-8 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-9 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-10 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-11 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-12 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-13 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-14 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-15 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-16 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
- Elute with 10ul Elution buffer
- Use 1ul for Qubit ssDNA assay
Qubit ssDNA assay results[edit]
- I did many bisulfite conversion reactions to compare the recovery rate with different inputs
Sample
|
Conc. in the Qubit
|
Unit
|
uL used
|
Dilution
|
Sample conc. (ng/ul)
|
Yield in 9ul (ng)
|
Input DNA (ng)
|
% Recovery
|
JK-1 |
3.8 |
ng/mL |
1 |
200 |
0.760 |
6.84 |
25 |
27.36
|
JK-2 |
4.9 |
ng/mL |
1 |
200 |
0.980 |
8.82 |
25 |
35.28
|
JK-3 |
2.5 |
ng/mL |
1 |
200 |
0.500 |
4.50 |
25 |
18.00
|
JK-4 |
5.11 |
ng/mL |
1 |
200 |
1.022 |
9.20 |
25 |
36.79
|
JK-5 |
10.8 |
ng/mL |
1 |
200 |
2.160 |
19.44 |
50 |
38.88
|
JK-6 |
9.06 |
ng/mL |
1 |
200 |
1.812 |
16.31 |
50 |
32.62
|
JK-7 |
12.3 |
ng/mL |
1 |
200 |
2.460 |
22.14 |
50 |
44.28
|
JK-8 |
11.2 |
ng/mL |
1 |
200 |
2.240 |
20.16 |
50 |
40.32
|
JK-9 |
25 |
ng/mL |
1 |
200 |
5.000 |
45.00 |
100 |
45.00
|
JK-10 |
26.7 |
ng/mL |
1 |
200 |
5.340 |
48.06 |
100 |
48.06
|
JK-11 |
28.4 |
ng/mL |
1 |
200 |
5.680 |
51.12 |
100 |
51.12
|
JK-12 |
24.3 |
ng/mL |
1 |
200 |
4.860 |
43.74 |
100 |
43.74
|
JK-13 |
90.1 |
ng/mL |
1 |
200 |
18.020 |
162.18 |
200 |
81.09
|
JK-14 |
89 |
ng/mL |
1 |
200 |
17.800 |
160.20 |
200 |
80.10
|
JK-15 |
79.7 |
ng/mL |
1 |
200 |
15.940 |
143.46 |
200 |
71.73
|
JK-16 |
85.4 |
ng/mL |
1 |
200 |
17.080 |
153.72 |
200 |
76.86
|
BSPP capture set up[edit]
strip 1
|
5:500
|
5:500
|
10:500
|
10:500
|
25:500
|
25:500
|
50:500
|
50:500
|
BIS-DNA required (ng) |
5 |
5 |
10 |
10 |
25 |
25 |
50 |
50
|
JK # |
JK-1 |
JK-2 |
JK-5 |
JK-6 |
JK-9 |
JK-10 |
JK-13 |
JK-14
|
Conc. |
0.760 |
0.980 |
2.160 |
1.812 |
5.000 |
5.340 |
18.020 |
17.800
|
Volume (ul) |
6.58 |
5.10 |
4.63 |
5.52 |
5.00 |
4.68 |
2.77 |
2.81
|
H2O (ul) |
0.27 |
1.75 |
2.07 |
1.18 |
1.25 |
1.57 |
2.72 |
2.69
|
V1N3 amount (ng) |
0.11 |
0.11 |
0.22 |
0.22 |
0.55 |
0.55 |
1.10 |
1.10
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.15 |
0.15 |
0.30 |
0.30 |
0.75 |
0.75 |
1.50 |
1.50
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
strip 2
|
5:1000
|
5:1000
|
10:1000
|
10:1000
|
25:1000
|
25:1000
|
50:1000
|
50:1000
|
BIS-DNA required (ng) |
5 |
5 |
10 |
10 |
25 |
25 |
50 |
50
|
JK # |
JK-4 |
JK-5 |
JK-7 |
JK-8 |
JK-11 |
JK-12 |
JK-15 |
JK-16
|
Conc. |
1.022 |
2.160 |
2.460 |
2.240 |
5.680 |
4.860 |
15.940 |
17.080
|
Volume (ul) |
4.89 |
2.31 |
4.07 |
4.46 |
4.40 |
5.14 |
3.14 |
2.93
|
H2O (ul) |
1.81 |
4.38 |
2.33 |
1.93 |
1.09 |
0.35 |
0.85 |
1.06
|
V1N3 amount (ng) |
0.22 |
0.22 |
0.44 |
0.44 |
1.10 |
1.10 |
2.20 |
2.20
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.30 |
0.30 |
0.60 |
0.60 |
1.50 |
1.50 |
3.01 |
3.01
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
strip 3
|
NTC:500
|
NTC:1000
|
BIS-DNA required (ng) |
0 |
0
|
JK # |
0 |
0
|
Conc. |
0 |
0
|
Volume (ul) |
0.00 |
0.00
|
H2O (ul) |
5.50 |
3.99
|
V1N3 amount (ng) |
1.10 |
2.20
|
V1N3 conc. (ng/ul) |
0.73 |
0.73
|
V1N3 volume (ul) |
1.50 |
3.01
|
10X Ampligase Buffer |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00
|
Total |
10.00 |
10.00
|
V1N3 & Ampligase Buffer mix
Mix 0.11
|
1x
|
4x
|
Diluted V1N3 |
0.15 |
0.60
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.15 |
|
Mix 0.22
|
1x
|
6x
|
Diluted V1N3 |
0.30 |
1.80
|
10X Ampligase Buffer |
1.00 |
6.00
|
H2O |
2.00 |
12.00
|
Sum |
3.30 |
|
Mix 0.44
|
1x
|
4x
|
Diluted V1N3 |
0.60 |
2.40
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.60 |
|
Mix 0.55
|
1x
|
4x
|
Diluted V1N3 |
0.75 |
3.00
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.75 |
|
Mix 1.1
|
1x
|
6x
|
Diluted V1N3 |
1.50 |
9.00
|
10X Ampligase Buffer |
1.00 |
6.00
|
H2O |
2.00 |
12.00
|
Sum |
4.50 |
|
Mix 2.2
|
1x
|
4x
|
Diluted V1N3 |
3.01 |
12.04
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
6.01 |
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 4:40h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold.
Amplification[edit]
- I used 10ul of captured DNA in total 75ul PCR reaction.
Components
|
1x rxn
|
20 rxn mix
|
Captured template |
10.00 |
0.00
|
10uM AmpF6.4Sol |
1.50 |
30.00
|
10uM AmpR6.3.IndX (X=57-72) |
1.50 |
0.00
|
2X KAPA SYBR MM |
37.50 |
750.00
|
H2O |
24.50 |
490.00
|
Total |
75.00 |
|
- - Aliquot 63.5ul + 1.5ul AmpR6.3 IndX + 10ul template
- - Set cycle number maximum at 27X
Program (Eppendorf Realplex)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min
Sample
|
Index
|
Sample
|
Index
|
500:1_5ng_1 |
Ind_57 |
1000:1_5ng_1 |
Ind_65
|
500:1_5ng_2 |
Ind_58 |
1000:1_5ng_2 |
Ind_66
|
500:1_10ng_1 |
Ind_59 |
1000:1_10ng_1 |
Ind_67
|
500:1_10ng_2 |
Ind_60 |
1000:1_10ng_2 |
Ind_68
|
500:1_25ng_1 |
Ind_61 |
1000:1_25ng_1 |
Ind_69
|
500:1_25ng_2 |
Ind_62 |
1000:1_25ng_2 |
Ind_70
|
500:1_50ng_1 |
Ind_63 |
1000:1_50ng_1 |
Ind_71
|
500:1_50ng_2 |
Ind_64 |
1000:1_50ng_2 |
Ind_72
|
PAGE verification[edit]
- Loaded 4ul of PCR products in 6% TBE gel
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 500to1.jpg
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 1000to1.jpg
- Dr. Zhang suggested to do sequencing on 5ng and 10ng input.
- I pooled PCR product by using the gel image intensity to get equimolar ratio of each libraries, including 5ng-1000:1 rep 1 &2 and 10ng-1000:1 rep1 and 2.
- Purified pooled PCR products with 0.8X AMPure beads and eluted with 40ul EB buffer.
- Loaded in 2-wells of 5-well 6% TBE gel
File:ZhangLab 2 2014-06-06 09hr 53min PAGE-SS V1N3-5-10ng.jpg
- Resuspend sequencing libraries in 20ul H2O
Sequencing library ID: NP-BSPP-MNV1N3-5-10ng, Jun1, 2014
- - Conc. by Qubit dsDNA HS = 0.644ng/ul (Alan did not have enough room for more libraries in qPCR)
- - Run: HL188 (required ~300k/library)
- - Length: 375bp
- - Index: 65-68 (5ng-rep1, 5ng-rep2, 10ng-rep1, 10ng-rep2)
- - They all used probe:target = 1000:1
Index
|
Sample
|
Ind_65 |
JK_5ng_1
|
Ind_66 |
JK_5ng_2
|
Ind_67 |
JK_10ng_1
|
Ind_68 |
JK_10ng_2
|
- - JK = Jurkat DNA