2023-04-11 E5 Sites 2 & 3 Species ID (POC and POR) via PCR and Sanger Sequencing

E5 Species ID (POC and POR) via PCR and Sanger Sequencing: Sites 2 and 3

Link with sample list and metadata

See base protocol and notes from Hollie

Followed my PCR Protocol exactly, based on instruction from Kristina Terpis

Sample List: All POC and POR colonies from the E5 timeseries need to be ID’d for one timepoint per colony. Starting with Site 2 and 3. Almost all samples are from Time Point 2 unless otherwise noted in the metadata (because no molecular sample was taken at TP2).

Amplification of 30 POC samples with mtORF on 4/11/2023.

n. timepoint collection_site colony_id Extraction Date Tech Primerset PCR Date
81 TP1 site2 POC-217 20211015 KXT mtORF 4/11/2023
267 TP2 site2 POC-257 20211112 KXT mtORF 4/11/2023
273 TP2 site2 POC-200 20211007 KXT mtORF 4/11/2023
289 TP2 site2 POC-205 20210924 KXT mtORF 4/11/2023
299 TP2 site2 POC-255 20211004 KXT mtORF 4/11/2023
305 TP2 site2 POC-222 20210923 KXT mtORF 4/11/2023
309 TP2 site2 POC-207 20211105 KXT mtORF 4/11/2023
311 TP2 site2 POC-254 20211019 KXT mtORF 4/11/2023
315 TP2 site2 POC-219 20210910 KXT mtORF 4/11/2023
317 TP2 site2 POC-239 20211104 KXT mtORF 4/11/2023
321 TP2 site2 POC-238 20211018 KXT mtORF 4/11/2023
323 TP2 site2 POC-259 20211108 KXT mtORF 4/11/2023
325 TP2 site2 POC-248 20211007 KXT mtORF 4/11/2023
329 TP2 site2 POC-201 20210930 KXT mtORF 4/11/2023
335 TP2 site2 POC-215 20211115 KXT mtORF 4/11/2023
345 TP2 site3 POC-371 20210907 KXT mtORF 4/11/2023
349 TP2 site3 POC-372 20210831 KXT mtORF 4/11/2023
355 TP2 site3 POC-373 20210920 KXT mtORF 4/11/2023
363 TP2 site3 POC-366 20210916 KXT mtORF 4/11/2023
367 TP2 site3 POC-391 20211109 KXT mtORF 4/11/2023
369 TP2 site3 POC-394 20210927 KXT mtORF 4/11/2023
373 TP2 site3 POC-359 20211108 KXT mtORF 4/11/2023
377 TP2 site3 POC-346 20211115 KXT mtORF 4/11/2023
419 TP2 site3 POC-369 20211101 KXT mtORF 4/11/2023
437 TP2 site3 POC-358 20211014 KXT mtORF 4/11/2023
447 TP2 site3 POC-395 20211122 KXT mtORF 4/11/2023
449 TP2 site3 POC-377 20211129 KXT mtORF 4/11/2023
455 TP2 site3 POC-375 20210921 KXT mtORF 4/11/2023
457 TP2 site3 POC-378 20211008 KXT mtORF 4/11/2023
465 TP2 site3 POC-386 20211005 KXT mtORF 4/11/2023

Positive control: Kristina’s E5 extracted DNA Tube #383 (POC 45).

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

2023-04-11-gel.JPG

Qubit Values:

n. Qubit AVG Nanodrop
81 17.65 121.00
267 17.20 125.90
273 17.00 131.40
289 15.60 118.60
299 16.15 123.00
305 16.40 119.00
309 16.15 117.90
311 16.90 115.40
315 19.65 129.10
317 16.70 134.80
321 17.10 132.20
323 16.30 125.40
325 18.25 132.20
329 18.15 132.40
335 19.55 143.70
345 15.20 126.50
349 15.90 128.90
355 14.00 135.80
363 17.50 126.70
367 13.55 128.90
369 17.50 131.20
373 17.50 130.90
377 14.15 99.20
419 18.95 119.60
437 11.85 88.00
447 11.85 96.90
449 17.40 117.50
455 13.90 105.90
457 13.35 106.90
465 13.15 92.80

SET UP for sequencing USING QUBIT VALUES (did not have access to nanodrop at the time, went back later and got nanodrop values), so did not dilute the PCR product.

Sample ID Well Template A. Template size B. Template stock conc. (ng/ul) C. PCR template: ng needed D. Volume=(C/B)ul E F. Volume PCR H20 needed (10-D or E) G. Primer Vol Sample # Colony
1   PCR 1000 17.65 25 1.4   8.6 2 81 POC-217
2   PCR 1000 17.65 25 1.4   8.6 2 81 POC-217
3   PCR 1000 17.20 25 1.5   8.5 2 267 POC-257
4   PCR 1000 17.20 25 1.5   8.5 2 267 POC-257
5   PCR 1000 17.00 25 1.5   8.5 2 273 POC-200
6   PCR 1000 17.00 25 1.5   8.5 2 273 POC-200
7   PCR 1000 15.60 25 1.6   8.4 2 289 POC-205
8   PCR 1000 15.60 25 1.6   8.4 2 289 POC-205
9   PCR 1000 16.15 25 1.5   8.5 2 299 POC-255
10   PCR 1000 16.15 25 1.5   8.5 2 299 POC-255
11   PCR 1000 16.40 25 1.5   8.5 2 305 POC-222
12   PCR 1000 16.40 25 1.5   8.5 2 305 POC-222
13   PCR 1000 16.15 25 1.5   8.5 2 309 POC-207
14   PCR 1000 16.15 25 1.5   8.5 2 309 POC-207
15   PCR 1000 16.90 25 1.5   8.5 2 311 POC-254
16   PCR 1000 16.90 25 1.5   8.5 2 311 POC-254
17   PCR 1000 19.65 25 1.3   8.7 2 315 POC-219
18   PCR 1000 19.65 25 1.3   8.7 2 315 POC-219
19   PCR 1000 16.70 25 1.5   8.5 2 317 POC-239
20   PCR 1000 16.70 25 1.5   8.5 2 317 POC-239
21   PCR 1000 17.10 25 1.5   8.5 2 321 POC-238
22   PCR 1000 17.10 25 1.5   8.5 2 321 POC-238
23   PCR 1000 16.30 25 1.5   8.5 2 323 POC-259
24   PCR 1000 16.30 25 1.5   8.5 2 323 POC-259
25   PCR 1000 18.25 25 1.4   8.6 2 325 POC-248
26   PCR 1000 18.25 25 1.4   8.6 2 325 POC-248
27   PCR 1000 18.15 25 1.4   8.6 2 329 POC-201
28   PCR 1000 18.15 25 1.4   8.6 2 329 POC-201
29   PCR 1000 19.55 25 1.3   8.7 2 335 POC-215
30   PCR 1000 19.55 25 1.3   8.7 2 335 POC-215
31   PCR 1000 15.20 25 1.6   8.4 2 345 POC-371
32   PCR 1000 15.20 25 1.6   8.4 2 345 POC-371
33   PCR 1000 15.90 25 1.6   8.4 2 349 POC-372
34   PCR 1000 15.90 25 1.6   8.4 2 349 POC-372
35   PCR 1000 14.00 25 1.8   8.2 2 355 POC-373
36   PCR 1000 14.00 25 1.8   8.2 2 355 POC-373
37   PCR 1000 17.50 25 1.4   8.6 2 363 POC-366
38   PCR 1000 17.50 25 1.4   8.6 2 363 POC-366
39   PCR 1000 13.55 25 1.8   8.2 2 367 POC-391
40   PCR 1000 13.55 25 1.8   8.2 2 367 POC-391
41   PCR 1000 17.50 25 1.4   8.6 2 369 POC-394
42   PCR 1000 17.50 25 1.4   8.6 2 369 POC-394
43   PCR 1000 17.50 25 1.4   8.6 2 373 POC-359
44   PCR 1000 17.50 25 1.4   8.6 2 373 POC-359
45   PCR 1000 14.15 25 1.8   8.2 2 377 POC-346
46   PCR 1000 14.15 25 1.8   8.2 2 377 POC-346
47   PCR 1000 18.95 25 1.3   8.7 2 419 POC-369
48   PCR 1000 18.95 25 1.3   8.7 2 419 POC-369
49   PCR 1000 11.85 25 2.1   7.9 2 437 POC-358
50   PCR 1000 11.85 25 2.1   7.9 2 437 POC-358
51   PCR 1000 11.85 25 2.1   7.9 2 447 POC-395
52   PCR 1000 11.85 25 2.1   7.9 2 447 POC-395
53   PCR 1000 17.40 25 1.4   8.6 2 449 POC-377
54   PCR 1000 17.40 25 1.4   8.6 2 449 POC-377
55   PCR 1000 13.90 25 1.8   8.2 2 455 POC-375
56   PCR 1000 13.90 25 1.8   8.2 2 455 POC-375
57   PCR 1000 13.35 25 1.9   8.1 2 457 POC-378
58   PCR 1000 13.35 25 1.9   8.1 2 457 POC-378
59   PCR 1000 13.15 25 1.9   8.1 2 465 POC-386
60   PCR 1000 13.15 25 1.9   8.1 2 465 POC-386

Amplification of 30 POR samples on 4/13/2023.

n. timepoint collection_site colony_id Extraction Date Tech Primerset PCR Date
269 TP2 site2 POR-221 20211102 KXT H2 4/13/2023
271 TP2 site2 POR-253 20211112 KXT H2 4/13/2023
275 TP2 site2 POR-240 20211007 KXT H2 4/13/2023
279 TP2 site2 POR-206/POR-251 20211115 KXT H2 4/13/2023
281 TP2 site2 POR-216 20211004 KXT H2 4/13/2023
283 TP2 site2 POR-209 20211108 KXT H2 4/13/2023
285 TP2 site2 POR-266 20211112 KXT H2 4/13/2023
287 TP2 site2 POR-235 20220201 KXT H2 4/13/2023
293 TP2 site2 POR-224 20211105 KXT H2 4/13/2023
297 TP2 site2 POR-245 20210924 KXT H2 4/13/2023
313 TP2 site2 POR-214 20210907 KXT H2 4/13/2023
319 TP2 site2 POR-236 20210923 KXT H2 4/13/2023
327 TP2 site2 POR-260 20210930 KXT H2 4/13/2023
333 TP2 site2 POR-242 20210913 KXT H2 4/13/2023
351 TP2 site3 POR-357 20211019 KXT H2 4/13/2023
353 TP2 site3 POR-353 20210831 KXT H2 4/13/2023
357 TP2 site3 POR-341 20210916 KXT H2 4/13/2023
361 TP2 site3 POR-355 20210913 KXT H2 4/13/2023
371 TP2 site3 POR-338 20210913 KXT H2 4/13/2023
375 TP2 site3 POR-349 20210920 KXT H2 4/13/2023
379 TP2 site3 POR-383 20211012 KXT H2 4/13/2023
381 TP2 site3 POR-365 20211001 KXT H2 4/13/2023
409 TP2 site3 POR-340 20210903 KXT H2 4/13/2023
445 TP2 site3 POR-367 20211018 KXT H2 4/13/2023
451 TP2 site3 POR-384 20211005 KXT H2 4/13/2023
453 TP2 site3 POR-354 20211028 KXT H2 4/13/2023
459 TP2 site3 POR-385 20220208 KXT H2 4/13/2023
461 TP2 site3 POR-381 20211118 KXT H2 4/13/2023
707 TP3 site3 POR-362 20211020 KXT H2 4/13/2023
877 TP4 site2 POR-262 20211018 KXT H2 4/13/2023

Positive control: Kristina’s E5 extracted DNA Tube #469 (extraction date: 20220208, PCR’d by me successfully in this post).

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

2023-04-13-gel.JPG

Did not clean up the double banding samples:

313 TP2 site2 POR-214

409 TP2 site3 POR-340

Nanodrop values

n. Nanodrop
269 99.20
271 103.80
275 141.50
279 155.50
281 143.70
283 146.90
285 138.60
287 99.90
293 113.50
297 154.30
313 Double banded
319 149.20
327 109.80
333 110.30
351 129.80
353 153.30
357 129.90
361 133.40
371 95.40
375 135.50
379 130.00
381 153.30
409 Double banded
445 144.70
451 122.80
453 106.70
459 114.50
461 124.70
707 61.80
877 115.90

SET UP the 28 that did not have double banding for sequencing 1:10th dilutions from nanodrop concentration

Sample ID Well Template A. Template size B. Template stock conc. (ng/ul) C. PCR template: ng needed D. Volume=(C/B)ul E F. Volume PCR H20 needed (10-D or E) G. Primer Vol Sample # Colony Undiluted nanodrop concentration
69   PCR 1500 9.92 37.5 3.8   6.2 2 269 POR-221 99.20
70   PCR 1500 9.92 37.5 3.8   6.2 2 269 POR-221 99.20
71   PCR 1500 10.38 37.5 3.6   6.4 2 271 POR-253 103.80
72   PCR 1500 10.38 37.5 3.6   6.4 2 271 POR-253 103.80
73   PCR 1500 14.15 37.5 2.7   7.3 2 275 POR-240 141.50
74   PCR 1500 14.15 37.5 2.7   7.3 2 275 POR-240 141.50
75   PCR 1500 15.55 37.5 2.4   7.6 2 279 POR-206/POR-251 155.50
76   PCR 1500 15.55 37.5 2.4   7.6 2 279 POR-206/POR-251 155.50
77   PCR 1500 14.37 37.5 2.6   7.4 2 281 POR-216 143.70
78   PCR 1500 14.37 37.5 2.6   7.4 2 281 POR-216 143.70
79   PCR 1500 14.69 37.5 2.6   7.4 2 283 POR-209 146.90
80   PCR 1500 14.69 37.5 2.6   7.4 2 283 POR-209 146.90
81   PCR 1500 13.86 37.5 2.7   7.3 2 285 POR-266 138.60
82   PCR 1500 13.86 37.5 2.7   7.3 2 285 POR-266 138.60
83   PCR 1500 9.99 37.5 3.8   6.2 2 287 POR-235 99.90
84   PCR 1500 9.99 37.5 3.8   6.2 2 287 POR-235 99.90
85   PCR 1500 11.35 37.5 3.3   6.7 2 293 POR-224 113.50
86   PCR 1500 11.35 37.5 3.3   6.7 2 293 POR-224 113.50
87   PCR 1500 15.43 37.5 2.4   7.6 2 297 POR-245 154.30
88   PCR 1500 15.43 37.5 2.4   7.6 2 297 POR-245 154.30
89   PCR 1500 14.92 37.5 2.5   7.5 2 319 POR-236 149.20
90   PCR 1500 14.92 37.5 2.5   7.5 2 319 POR-236 149.20
91   PCR 1500 10.98 37.5 3.4   6.6 2 327 POR-260 109.80
92   PCR 1500 10.98 37.5 3.4   6.6 2 327 POR-260 109.80
93   PCR 1500 11.03 37.5 3.4   6.6 2 333 POR-242 110.30
94   PCR 1500 11.03 37.5 3.4   6.6 2 333 POR-242 110.30
95   PCR 1500 12.98 37.5 2.9   7.1 2 351 POR-357 129.80
96   PCR 1500 12.98 37.5 2.9   7.1 2 351 POR-357 129.80
97   PCR 1500 15.33 37.5 2.4   7.6 2 353 POR-353 153.30
98   PCR 1500 15.33 37.5 2.4   7.6 2 353 POR-353 153.30
99   PCR 1500 12.99 37.5 2.9   7.1 2 357 POR-341 129.90
100   PCR 1500 12.99 37.5 2.9   7.1 2 357 POR-341 129.90
101   PCR 1500 13.34 37.5 2.8   7.2 2 361 POR-355 133.40
102   PCR 1500 13.34 37.5 2.8   7.2 2 361 POR-355 133.40
103   PCR 1500 9.54 37.5 3.9   6.1 2 371 POR-338 95.40
104   PCR 1500 9.54 37.5 3.9   6.1 2 371 POR-338 95.40
105   PCR 1500 13.55 37.5 2.8   7.2 2 375 POR-349 135.50
106   PCR 1500 13.55 37.5 2.8   7.2 2 375 POR-349 135.50
107   PCR 1500 13.00 37.5 2.9   7.1 2 379 POR-383 130.00
108   PCR 1500 13.00 37.5 2.9   7.1 2 379 POR-383 130.00
109   PCR 1500 15.33 37.5 2.4   7.6 2 381 POR-365 153.30
110   PCR 1500 15.33 37.5 2.4   7.6 2 381 POR-365 153.30
111   PCR 1500 14.47 37.5 2.6   7.4 2 445 POR-367 144.70
112   PCR 1500 14.47 37.5 2.6   7.4 2 445 POR-367 144.70
113   PCR 1500 12.28 37.5 3.1   6.9 2 451 POR-384 122.80
114   PCR 1500 12.28 37.5 3.1   6.9 2 451 POR-384 122.80
115   PCR 1500 10.67 37.5 3.5   6.5 2 453 POR-354 106.70
116   PCR 1500 10.67 37.5 3.5   6.5 2 453 POR-354 106.70
117   PCR 1500 11.45 37.5 3.3   6.7 2 459 POR-385 114.50
118   PCR 1500 11.45 37.5 3.3   6.7 2 459 POR-385 114.50
119   PCR 1500 12.47 37.5 3.0   7.0 2 461 POR-381 124.70
120   PCR 1500 12.47 37.5 3.0   7.0 2 461 POR-381 124.70
121   PCR 1500 6.18 37.5 6.1   3.9 2 707 POR-362 61.80
122   PCR 1500 6.18 37.5 6.1   3.9 2 707 POR-362 61.80
123   PCR 1500 11.59 37.5 3.2   6.8 2 877 POR-262 115.90
124   PCR 1500 11.59 37.5 3.2   6.8 2 877 POR-262 115.90

POC mtORF (ZD01-8) test samples were sent For Sanger Sequencing on 4/13/2023. The rest of the POC mtORF (ZD09-68) and POR H2 (ZD69-ZD124) (except for the 2 samples with double banding) will be sent for sequencing on 4/18/2023.

Double Banding in POR H2

These samples had double banding in the gel on 4/13/23

313 TP2 site2 POR-214

409 TP2 site3 POR-340

To test if the double banding was due to poor DNA quality, I re-PCRd these colonies from different time points:

27 TP1 site2 POR-214

605 TP3 site3 POR-340

Positive control: Kristina’s E5 extracted DNA Tube #371 (extraction date: 20210913, PCR’d by me successfully above).

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

2023-04-17-gel.JPG

Next steps: run large comb gel with the rest of the PCR product and perform a gell extraction on the band that has the same size as the positive control ~1500bp. Need UV protection.

2023-04-20-gel-1.JPG

Excise bands at same length as positive control:

2023-04-20-gel-2.JPG

Perform cleanup of excised bands using Zymoclean Kit

(Will write this up ASAP, for now see notebook page images here and here)

Run second gel to quality check and confirm only one band is remaining in the cleaned up product.

2023-04-20-gel-3.JPG

Set up for sequencing using nanodrop values (no dilution needed), will be sequenced on 4/24/23.

Sample ID Well Template A. Template size B. Template stock conc. (ng/ul) C. PCR template: ng needed D. Volume=(C/B)ul E F. Volume PCR H20 needed (10-D or E) G. Primer Vol Sample # Colony
125   PCR 1500 32.10 37.5 1.2   8.8 2 27 POR-214
126   PCR 1500 32.10 37.5 1.2   8.8 2 27 POR-214
127   PCR 1500 40.40 37.5 0.9   9.1 2 605 POR-340
128   PCR 1500 40.40 37.5 0.9   9.1 2 605 POR-340
Written on April 11, 2023