QuantSeq Library Generation Batch 2
Generated libraries on my second batch of ctenidia RNA samples.
Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.
Sample order in plate |
Sample No. |
[RNA] (ng/ul) |
Vol RNA used |
Vol H2O to add |
ng RNA used |
1 |
343 |
114.0 |
3.07 |
1.93 |
350 |
2 |
345 |
190.0 |
1.84 |
3.16 |
350 |
3 |
303 |
95.2 |
3.68 |
1.32 |
350 |
4 |
346 |
43.6 |
5.00 |
- |
218 |
5 |
302 |
62.4 |
5.00 |
- |
312 |
6 |
336 |
94.8 |
3.69 |
1.31 |
350 |
7 |
292 |
29.6 |
5.00 |
- |
148 |
8 |
NTC1 -B2 |
- |
- |
5.00 |
0 |
9 |
317 |
158.0 |
2.22 |
2.78 |
350 |
10 |
322 |
44.6 |
5.00 |
- |
223 |
11 |
332 |
65.8 |
5.00 |
- |
329 |
12 |
334 |
64.8 |
5.00 |
- |
324 |
13 |
349 |
82.0 |
4.27 |
0.73 |
350 |
14 |
337 |
194.0 |
1.80 |
3.20 |
350 |
15 |
341 |
89.6 |
3.91 |
1.09 |
350 |
16 |
313 |
72.4 |
4.83 |
0.17 |
350 |
17 |
309 |
170.0 |
2.06 |
2.94 |
350 |
18 |
327 |
85.2 |
4.11 |
0.89 |
350 |
19 |
319 |
77.6 |
4.51 |
0.49 |
350 |
20 |
326 |
130.0 |
2.69 |
2.31 |
350 |
21 |
306 |
136.0 |
2.57 |
2.43 |
350 |
22 |
323 |
102.0 |
3.43 |
1.57 |
350 |
23 |
314 |
42.2 |
5.00 |
- |
211 |
24 |
316 |
146.0 |
2.40 |
2.60 |
350 |
25 |
339 |
77.2 |
4.53 |
0.47 |
350 |
26 |
293 |
39.6 |
5.00 |
- |
198 |
27 |
329 |
162.0 |
2.16 |
2.84 |
350 |
28 |
296 |
180.0 |
1.94 |
3.06 |
350 |
Volumes of solutions needed - I aliquoted volumes into 7 pcr tubes, so I could then add to samples using a multichannel pipette.
Step, Chem. |
Vol per rxn (uL) |
Total + 10% or 15% |
Vol per aliquot (n=7) |
Vol per sample |
Step 3 MM: FS2 |
9.5 |
305.9 |
46 |
10 |
Step 3 MM: E1 |
0.5 |
16.1 |
|
|
Step 6: RS |
5 |
154 |
22 |
5 |
Step 7: SS1 |
10 |
308 |
44 |
10 |
Step 9 MM: SS2 |
4 |
128.8 |
23 |
5 |
Step 9 MM: E2 |
1 |
32.2 |
|
|
PCR Plate setup
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
A |
343 |
345 |
303 |
346 |
302 |
336 |
292 |
|
B |
|
|
|
|
|
|
|
|
C |
NTC1 -B2 |
317 |
322 |
332 |
334 |
349 |
337 |
|
D |
|
|
|
|
|
|
|
|
E |
341 |
313 |
309 |
327 |
319 |
326 |
306 |
|
F |
|
|
|
|
|
|
|
|
G |
323 |
314 |
316 |
339 |
293 |
329 |
296 |
|
H |
|
|
|
|
|
|
|
|
Notes
- I worked in 4 rows of 7 (27 samples + 1 NTC).
- I accidentally used 4.51 uL of sample #326, which is ~585 ug of RNA (exceeds the 500 ug max). I proceeded anyway, and will see if it influences the library quality.
- Protocol was slightly improved by shortening the amount of time samples sit at 42C at step 2/3 (from 15 mins to ~7 minutes), and I cut the PCR plate to only have 8 of the 12 columns, which freed up space for pre-warming master mix #1 (steps 2/3).
- I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.
Written on December 6, 2019