QuantSeq Library Generation Batch 1
Began my full QuantSeq library prep today. I am processing ctenidia samples first, and since there are 53 samples I’m doing ~half at a time. Today I generated double stranded cDNA for 26 samples + 2 NTC (28 total). I loaded samples onto a PCR plate in 4 rows of 7.
Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.
cDNA synthesis |
12/5/19 |
# samples + 2 NTC |
28 |
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Work in 4 rows of 7 |
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Sample order in plate |
Sample No. |
[RNA] (ng/ul) |
Vol RNA used |
Vol H2O to add |
ng RNA used |
1 |
328 |
156.0 |
2.24 |
2.76 |
350 |
2 |
299 |
50.4 |
5.00 |
- |
252 |
3 |
301 |
75.8 |
4.62 |
0.38 |
350 |
4 |
342 |
162.0 |
2.16 |
2.84 |
350 |
5 |
331 |
42.2 |
5.00 |
- |
211 |
6 |
307 |
89.4 |
3.91 |
1.09 |
350 |
7 |
295 |
34.8 |
5.00 |
- |
174 |
8 |
304 |
200.0 |
1.75 |
3.25 |
350 |
9 |
305 |
75.2 |
4.65 |
0.35 |
350 |
10 |
311 |
158.0 |
2.22 |
2.78 |
350 |
11 |
NTC2 - B1 |
- |
- |
5.00 |
0 |
12 |
298 |
182.0 |
1.92 |
3.08 |
350 |
13 |
348 |
54.4 |
5.00 |
- |
272 |
14 |
315 |
148.0 |
2.36 |
2.64 |
350 |
15 |
344 |
25.0 |
5.00 |
- |
125 |
16 |
325 |
180.0 |
1.94 |
3.06 |
350 |
17 |
338 |
81.6 |
4.29 |
0.71 |
350 |
18 |
347 |
69.0 |
5.00 |
- |
345 |
19 |
312 |
90.6 |
3.86 |
1.14 |
350 |
20 |
321 |
148.0 |
2.36 |
2.64 |
350 |
21 |
333 |
78.6 |
4.45 |
0.55 |
350 |
22 |
291 |
158.0 |
2.22 |
2.78 |
350 |
23 |
308 |
73.6 |
4.76 |
0.24 |
350 |
24 |
NTC1 - B1 |
- |
- |
5.00 |
0 |
25 |
335 |
180.0 |
1.94 |
3.06 |
350 |
26 |
318 |
174.0 |
2.01 |
2.99 |
350 |
27 |
294 |
110.0 |
3.18 |
1.82 |
350 |
28 |
324 |
172.0 |
2.03 |
2.97 |
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 + 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 |
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Step 6: RS |
5 |
161 |
23 |
5 |
Step 7: SS1 |
10 |
322 |
46 |
10 |
Step 9 MM: SS2 |
4 |
128.8 |
23 |
5 |
Step 9 MM: E2 |
1 |
32.2 |
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PCR Plate setup
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
A |
328 |
299 |
301 |
342 |
331 |
307 |
295 |
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B |
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C |
304 |
305 |
311 |
NTC2 - B1 |
298 |
348 |
315 |
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D |
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E |
344 |
325 |
338 |
347 |
312 |
321 |
333 |
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F |
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G |
291 |
308 |
NTC1 - B1 |
335 |
318 |
294 |
324 |
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H |
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Notes
- Samples were thawed on wet ice, and vortexed once before use.
- I loaded the first 3 samples, 328, 299 and 301, onto the PCR plate but then had to wait a few minutes (~3-5) for the rest to thaw.
- Step #2: held samples at 42C for 14 minutes while preparing master mix. Probably a bit too long.
- I used a whole PCR Plate, which took up the entire thermocycler space. Next time, I should use partial PCR plates to leave room for a PCR strip to pre-warm the master mix needed for step 3 & 4.
- The centrifuge gradually cooled as I was using it, which was weird since I didn’t change the temp. I think I need to set temperature to 22C every time I use it, b/c it may default to 4C.
- I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.
Written on December 5, 2019