Revision as of 07:21, 16 June 2014 by >Noi
MONOD V1N3 BSPP capture of serum and solid tumor DNA
serum DNA
- Some samples had volume >20ul, I evaporated to reduce the volume (~from 30ul --> 13/14ul, 30C for 10min)
Sample
|
Original conc. (ng/ul)
|
Original vol. before evap.
|
Volume for 50ng (ul)
|
H2O (ul)
|
Lightning Conv. Reagent (ul)
|
Total volume (ul)
|
Total DNA amount (ng)
|
PC-S_2 (evap) |
1.66 |
27.00 |
14.00 |
6.00 |
130.00 |
150.00 |
44.82
|
PC-S_3 |
21.40 |
NA |
2.34 |
17.66 |
130.00 |
150.00 |
50.00
|
PC-S_5 (evap) |
1.58 |
27.00 |
14.00 |
6.00 |
130.00 |
150.00 |
42.66
|
PC-S_6 |
17.50 |
NA |
2.86 |
17.14 |
130.00 |
150.00 |
50.00
|
PC-S_7 (evap) |
1.80 |
25.00 |
14.00 |
6.00 |
130.00 |
150.00 |
45.00
|
PC-S_9 |
5.72 |
NA |
8.74 |
11.26 |
130.00 |
150.00 |
50.00
|
PC-S_10 (evap) |
1.71 |
27.00 |
13.00 |
7.00 |
130.00 |
150.00 |
46.17
|
Solid tumor tissue DNA
Sample
|
Conc. (ng/ul)
|
Volume for 50ng (ul)
|
H2O (ul)
|
Lightning Conv. Reagent (ul)
|
Total volume (ul)
|
Total DNA amount (ng)
|
PC-T-1 |
82.70 |
0.60 |
19.40 |
130.00 |
150.00 |
50.00
|
PC-T-2 |
35.80 |
1.40 |
18.60 |
130.00 |
150.00 |
50.00
|
PC-T-4 |
42.80 |
1.17 |
18.83 |
130.00 |
150.00 |
50.00
|
PC-T-6 |
15.20 |
3.29 |
16.71 |
130.00 |
150.00 |
50.00
|
PC-C-7 |
16.30 |
3.07 |
16.93 |
130.00 |
150.00 |
50.00
|
- I did the 2nd set for those samples with high amount of DNA. Just in case there is something wrong with the result of some samples.
Sample
|
Conc. (ng/ul)
|
Volume for 50ng (ul)
|
H2O (ul)
|
Lightning Conv. Reagent (ul)
|
Total volume (ul)
|
Total DNA amount (ng)
|
PC-S_3 |
21.40 |
2.34 |
17.66 |
130.00 |
150.00 |
50.00
|
PC-S_6 |
17.50 |
2.86 |
17.14 |
130.00 |
150.00 |
50.00
|
PC-S_9 |
5.72 |
8.74 |
11.26 |
130.00 |
150.00 |
50.00
|
PC-T-1 |
82.70 |
0.60 |
19.40 |
130.00 |
150.00 |
50.00
|
PC-T-2 |
35.80 |
1.40 |
18.60 |
130.00 |
150.00 |
50.00
|
PC-T-4 |
42.80 |
1.17 |
18.83 |
130.00 |
150.00 |
50.00
|
PC-T-6 |
15.20 |
3.29 |
16.71 |
130.00 |
150.00 |
50.00
|
PC-C-7 |
16.30 |
3.07 |
16.93 |
130.00 |
150.00 |
50.00
|
- Elute BIS-DNA with 9.2ul Elution buffer and expected DNA volume left ~8.4ul for capture, but I only have DNA left 7ul. Next time, I will elute with ~10ul Elution buffer.
BSPP capture set up
- Since the input DNA for BIS-CVT was quite low, I did not measure BIS-DNA concentration since I did not want to lose DNA. I assumed that there was DNA after BIS-CVT ~10ng from 20% recovery rate.
- I will use probe to target ration 1000:1 --> requires V1N3 amount 0.44ug from 10X diluted V1N3 (0.73ng/ul)
strip 1
|
PC-S_2
|
PC-S_3
|
PC-S_5
|
PC-S_6
|
PC-S_7
|
PC-S_9
|
PC-S_10
|
Volume (ul) |
7.00 |
7.00 |
7.00 |
7.00 |
7.00 |
7.00 |
7.00
|
V1N3 amount (ng) |
0.44 |
0.44 |
0.44 |
0.44 |
0.44 |
0.44 |
0.44
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.60 |
0.60 |
0.60 |
0.60 |
0.60 |
0.60 |
0.60
|
H2O in buffer mix |
1.40 |
1.40 |
1.40 |
1.40 |
1.40 |
1.40 |
1.40
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
strip 2
|
PC-T-1
|
PC-T-2
|
PC-T-4
|
PC-T-6
|
PC-C-7
|
NTC
|
Volume (ul) |
7.00 |
7.00 |
7.00 |
7.00 |
7.00 |
7.00
|
V1N3 amount (ng) |
0.44 |
0.44 |
0.44 |
0.44 |
0.44 |
0.44
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.60 |
0.60 |
0.60 |
0.60 |
0.60 |
0.60
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
H2O in buffer mix |
1.40 |
1.40 |
1.40 |
1.40 |
1.40 |
1.40
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
- With the same condition as samples above
PC-S_3
|
PC-S_6
|
PC-S_9
|
PC-T-1
|
PC-T-2
|
PC-T-4
|
PC-T-6
|
PC-C-7
|
V1N3 & 10X Ampligase Buffer Mix
Components
|
1x rxn
|
25x rxn
|
V1N3 (0.73ng/ul) |
0.60 |
15.00
|
10X Ampligase Buffer Mix |
1.00 |
25.00
|
H2O |
1.40 |
35.00
|
Total |
3.00 |
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 1ul KLN mix (2U/ul Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 6h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 5min -> 4C hold.
KLN Solution Mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume total 30ul
|
Hemo KlenTaq |
100 |
% |
20 |
% |
6.00
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
3.00
|
dNTP |
1 |
mM |
100 |
uM |
3.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
3.00
|
H2O |
|
|
|
|
15.00
|
Amplification
- Used the same condition for PCR as experiment on 2014-06-02 with 10ul captured DNA in total 75ul
Components
|
1x rxn
|
22.5 rxn mix
|
Captured template |
10.00 |
0.00
|
10uM AmpF6.4Sol |
1.50 |
33.75
|
10uM AmpR6.3.IndX |
1.50 |
0.00
|
2X KAPA SYBR MM |
37.50 |
843.75
|
H2O |
24.50 |
551.25
|
Total |
75.00 |
|
Program (Eppendorf Realplex)
95C 30s -> (95C 10s -> 58C 20s -> 72C 20s)x8 -> (95C 10s -> 72C 20s)x19 -> 72C 3min
quantification of individual sequencing libraries
V1N3-S
File:ZhangLab 2 2014-06-12 15hr 10min PC-S V1N3.jpg
I did not include index43 in the library pools as the concentration is too low.
Sample
|
Index
|
Conc. (ng/ul)
|
Yield in 35ul (ng)
|
Volume for 20ng (ul)
|
PC-S_2 |
Ind_41 |
2.26 |
79.04 |
8.86
|
PC-S_3 |
Ind_42 |
1.11 |
38.78 |
18.05
|
PC-S_6 |
Ind_44 |
0.71 |
24.86 |
28.16
|
PC-S_7 |
Ind_45 |
1.39 |
48.75 |
14.36
|
PC-S_9 |
Ind_46 |
0.75 |
26.14 |
26.78
|
PC-S_10 |
Ind_47 |
0.78 |
27.47 |
25.48
|
- Purified in 2 well of 5-well 6% TBE PAGE
V1N3-T
File:ZhangLab 2 2014-06-12 15hr 10min PC-T V1N3.jpg
Sample
|
Index
|
Conc. (ng/ul)
|
Yield in 35ul (ng)
|
Volume for 30ng (ul)
|
PC-T-1 |
Ind_51 |
2.99 |
104.81 |
10.02
|
PC-T-2 |
Ind_52 |
2.50 |
87.33 |
12.02
|
PC-T-4 |
Ind_53 |
2.96 |
103.45 |
10.15
|
PC-T-6 |
Ind_54 |
1.13 |
39.70 |
26.45
|
PC-C-7 |
Ind_55 |
2.99 |
104.62 |
10.04
|
- Purified in 2 well of 5-well 6% TBE PAGE
NP-BSPP-V1N3-S-Jun10, 2014 NP-BSPP-V1N3-T-Jun10, 2014
File:ZhangLab 2 2014-06-13 10hr 19min V1N3 serum PAGE-SS.jpg File:ZhangLab 2 2014-06-13 10hr 19min V1N3 tumor PAGE-SS.jpg
- Since we are going to get sequencing at Illumina, I pooled three library pools to one tube with the same molar ratio
Library pool
|
# of libraries
|
Ratio toV1N3-P
|
Conc.by Qubit dsDNA HS (ng/ul)
|
Pool 4ng of V1N3-P (ul)
|
V1N3-S |
6 |
1.500 |
0.590 |
10.169
|
V1N3-T |
5 |
1.250 |
0.610 |
8.197
|
V1N3-P |
4 |
1.000 |
0.488 |
8.197
|
Index for serum samples
- I used barcoded primers in column #6
strip 1
|
PC-S_2
|
PC-S_3
|
PC-S_5
|
PC-S_6
|
PC-S_7
|
PC-S_9
|
PC-S_10
|
Index |
Ind_41 |
Ind_42 |
Ind_43 |
Ind_44 |
Ind_45 |
Ind_46 |
Ind_47
|
- I used barcoded primers in column #7 and start at well C1 (= Ind_51) to avoid bad barcodes
Project ID
|
Sample type
|
Zhang lab ID
|
Index
|
UCSD-004-05 |
Serum |
PC-S_2 |
Ind_41
|
UCSD-004-06 |
Serum |
PC-S_3 |
Ind_42
|
UCSD-004-08 |
Serum |
PC-S_5 |
Ind_43
|
UCSD-004-09 |
Serum |
PC-S_6 |
Ind_44
|
UCSD-004-10 |
Serum |
PC-S_7 |
Ind_45
|
UCSD-004-12 |
Serum |
PC-S_9 |
Ind_46
|
UCSD-004-13 |
Serum |
PC-S_10 |
Ind_47
|
Index for solid tumor tissue samples
strip 2
|
PC-T-1
|
PC-T-2
|
PC-T-4
|
PC-T-6
|
PC-C-7
|
NTC
|
Index |
Ind_51 |
Ind_52 |
Ind_53 |
Ind_54 |
Ind_55 |
Ind_56
|
Project ID
|
Sample type
|
Zhang lab ID
|
Index
|
UCSD-004-04 |
Solid tumor tissue |
PC-T-1 |
Ind_51
|
UCSD-004-05 |
Solid tumor tissue |
PC-T-2 |
Ind_52
|
UCSD-004-07 |
Solid tumor tissue |
PC-T-4 |
Ind_53
|
UCSD-004-09 |
Solid tumor tissue |
PC-T-6 |
Ind_54
|
UCSD-004-10 |
Solid tumor tissue |
PC-C-7 |
Ind_55
|
Index for plasma sample from Kang Zhang's lab with pre-amplification
- Lab note on 2014-06-07: [[1]]
Index
|
Sample
|
Conc. (ng/ul)
|
Yield in 70ul (ng)
|
Volume for 40ng (ul)
|
Ind_33 |
NC-P-5ng_1 |
1.00 |
69.68 |
40.19
|
Ind_34 |
NC-P-5ng_2 |
0.62 |
43.70 |
50.00**
|
Ind_35 |
NC-P-10ng_1 |
0.88 |
61.38 |
45.62
|
Ind_36 |
NC-P-10ng_2 |
1.26 |
88.54 |
31.63
|