QuantSeq Library Generation Batches 3 and 4
Sample order in plate |
Sample No. |
[RNA] (ng/ul) |
Vol RNA used |
Vol H2O to add |
ng RNA used |
1 |
35 |
94.4 |
3.71 |
1.29 |
350 |
2 |
524 |
63.0 |
5.00 |
- |
315 |
3 |
453 |
196.0 |
1.79 |
3.21 |
350 |
4 |
554 |
188.0 |
1.86 |
3.14 |
350 |
5 |
442b |
69.8 |
5.00 |
- |
349 |
6 |
489 |
68.0 |
5.00 |
- |
340 |
7 |
462b |
106.0 |
3.30 |
1.70 |
350 |
8 |
533 |
6.5 |
5.00 |
- |
32.6 |
9 |
522 |
32.2 |
5.00 |
- |
161 |
10 |
474 |
77.2 |
4.53 |
0.47 |
350 |
11 |
452b |
97.2 |
3.60 |
1.40 |
350 |
12 |
443 |
60.2 |
5.00 |
- |
301 |
13 |
477 |
164.0 |
2.13 |
2.87 |
350 |
14 |
526 |
138.0 |
2.54 |
2.46 |
350 |
15 |
562 |
126.0 |
2.78 |
2.22 |
350 |
16 |
432 |
74.0 |
4.73 |
0.27 |
350 |
17 |
37 |
74.6 |
4.69 |
0.31 |
350 |
18 |
413 |
130.0 |
2.69 |
2.31 |
350 |
19 |
45 |
25.1 |
5.00 |
- |
125.5 |
20 |
561 |
28.0 |
5.00 |
- |
140 |
21 |
542 |
32.8 |
5.00 |
- |
164 |
22 |
527 |
124.0 |
2.82 |
2.18 |
350 |
23 |
492 |
82.6 |
4.24 |
0.76 |
350 |
24 |
475 |
27.4 |
5.00 |
- |
137 |
25 |
541 |
44.4 |
5.00 |
- |
222 |
26 |
565 |
31.2 |
5.00 |
- |
156 |
27 |
B3 - NTC1 |
NA |
- |
5.00 |
#VALUE! |
28 |
B3 - NTC2 |
NA |
- |
5.00 |
#VALUE! |
Batch 3 Plate Configuration
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
A |
35 |
524 |
453 |
554 |
442b |
489 |
462b |
|
B |
|
|
|
|
|
|
|
|
C |
533 |
522 |
474 |
452b |
443 |
477 |
526 |
|
D |
|
|
|
|
|
|
|
|
E |
562 |
432 |
37 |
413 |
45 |
561 |
542 |
|
F |
|
|
|
|
|
|
|
|
G |
527 |
492 |
475 |
541 |
565 |
B3 - NTC1 |
B3 - NTC2 |
|
H |
|
|
|
|
|
|
|
|
Sample order in plate |
Sample No. |
[RNA] (ng/ul) |
Vol RNA used |
Vol H2O to add |
ng RNA used |
1 |
571 |
LOW |
5.00 |
- |
NA |
2 |
525 |
140.0 |
2.50 |
2.50 |
350 |
3 |
563 |
47.0 |
5.00 |
- |
235 |
4 |
404 |
112.0 |
3.13 |
1.88 |
350 |
5 |
484 |
58.4 |
5.00 |
- |
292 |
6 |
531 |
95.4 |
3.67 |
1.33 |
350 |
7 |
34 |
104.0 |
3.37 |
1.63 |
350 |
8 |
490 |
186.0 |
1.88 |
3.12 |
350 |
9 |
523 |
71.4 |
4.90 |
0.10 |
350 |
10 |
473 |
124.0 |
2.82 |
2.18 |
350 |
11 |
485 |
19.1 |
5.00 |
- |
95.5 |
12 |
402 |
114.0 |
3.07 |
1.93 |
350 |
13 |
487 |
118.0 |
2.97 |
2.03 |
350 |
14 |
476 |
39.2 |
5.00 |
- |
196 |
15 |
421 |
57.6 |
5.00 |
- |
288 |
16 |
553 |
186.0 |
1.88 |
3.12 |
350 |
17 |
41 |
108.0 |
3.24 |
1.76 |
350 |
18 |
46 |
98.6 |
3.55 |
1.45 |
350 |
19 |
551 |
96.4 |
3.63 |
1.37 |
350 |
20 |
486b |
148.0 |
2.36 |
2.64 |
350 |
21 |
445 |
160.0 |
2.19 |
2.81 |
350 |
22 |
43 |
156.0 |
2.24 |
2.76 |
350 |
23 |
506 |
29.2 |
5.00 |
- |
146 |
24 |
482 |
22.2 |
5.00 |
- |
111 |
25 |
412 |
31.2 |
5.00 |
- |
156 |
26 |
488 |
60.0 |
5.00 |
- |
300 |
27 |
47 |
128.0 |
2.73 |
2.27 |
350 |
28 |
B4 - NTC2 |
NA |
- |
5.00 |
0 |
Batch 4 Plate Configuration
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
A |
571 |
525 |
563 |
404 |
484 |
531 |
34 |
|
B |
|
|
|
|
|
|
|
|
C |
490 |
523 |
473 |
485 |
402 |
487 |
476 |
|
D |
|
|
|
|
|
|
|
|
E |
421 |
553 |
41 |
46 |
551 |
486b |
445 |
|
F |
|
|
|
|
|
|
|
|
G |
43 |
506 |
482 |
412 |
488 |
47 |
B4 - NTC2 |
|
H |
|
|
|
|
|
|
|
|
Notes
- Aliquoted 20 uL RNA into new tubes and froze in -80. Except, for the following samples I aliquoted 7 uL: 34, 35, 41, 46, 46
- Sample #35 was not fully sealed during either the RNA removal step or the second strand synthesis step, and I believe I lost some volume to evaporation. I proceeded normally, but will see how that library looks.
- Not sure if I mentioned this previously, but I’m using 350 ng RNA, except for low concentration samples (I use 5 uL to get the maximum amount of RNA possible).
- To “quickly spin down” samples at room temperature I put the sample plate in the centrifuge, push start, then push stop immediately when the speed does not read 0. I also set the temperature to room temperature so it doesn’t actively cool.
Written on December 14, 2019