Laura’s Notebook: 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 Work in 4 rows of 7
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
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

PCR Plate setup

1 2 3 4 5 6 7 8 9 10 11 12
A 328 299 301 342 331 307 295
B
C 304 305 311 NTC2 – B1 298 348 315
D
E 344 325 338 347 312 321 333
F
G 291 308 NTC1 – B1 335 318 294 324
H

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.

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