mtORF Amplification for Pocillopora spp. corals

Amplification of the mitochondria open reading frame (mtORF) region from DNA extracted from Pocillopora coral fragments from the Holobiont Integration 2018 project.

Following the Putnam Lab’s mtORF Amplification and Sanger Sequencing Prep Protocol.

Master spreadsheet for Holobiont Integration mtORF samples here.

10 ng/uL Dilutions

Each sample is diluted to 10 ng/uL concentration to standardize how much DNA we start with prior to PCR reaction.

Plug_ID	Extraction Date	Species	Hard DNA (ng_ul)	DNA for dilution (ul)	Water for dilution (ul)	Notes
1041	20190823	Pocillopora	44	2.27	7.73	✓
1043	20190814	Pocillopora	25.9	3.86	6.14	✓
1047	20190808	Pocillopora	37.4	2.67	7.33	✓
1050	20190809	Pocillopora	35.4	2.82	7.18	✓
1051	20190731	Pocillopora	45.5	2.20	7.80	✓
1059	20190826	Pocillopora	25.2	3.97	6.03	✓
1060	20190725	Pocillopora	44.3	2.26	7.74	✓
1090	20190807	Pocillopora	14	7.14	2.86	✓
1103	20190718	Pocillopora	50.9	1.96	8.04	✓
1131	20190718	Pocillopora	30.5	3.28	6.72	✓
1138	20190809	Pocillopora	33.1	3.02	6.98	✓
1141	20190722	Pocillopora	24.1	4.15	5.85	✓
1147	20191206	Pocillopora	39.3	2.54	7.46	✓
1159	20190720	Pocillopora	4.38	22.83	-12.83	✓
1168	20190730	Pocillopora	21.5	4.65	5.35	✓
1184	20190731	Pocillopora	33.7	2.97	7.03	✓
1205	20190720	Pocillopora	27.3	3.66	6.34	✓
1207	20190826	Pocillopora	35.9	2.79	7.21	✓
1219	20190724	Pocillopora	40.2	2.49	7.51	✓
1220	20190730	Pocillopora	20.3	4.93	5.07	✓
1225	20190730	Pocillopora	21.6	4.63	5.37	✓
1227	20190801	Pocillopora	10.55	9.48	0.52	✓
1238	20190725	Pocillopora	27.4	3.65	6.35	✓
1239	20190823	Pocillopora	28.6	3.50	6.50	✓
1254	20190731	Pocillopora	32.7	3.06	6.94	✓
1281	20190924	Pocillopora	16.7	5.99	4.01	✓
1296	20190729	Pocillopora	21.5	4.65	5.35	✓
1302	20191111	Pocillopora	15.55	6.43	3.57	✓
1303	20190726	Pocillopora	18.85	5.31	4.69	✓
1329	20191204	Pocillopora	48.1	2.08	7.92	✓
1330	20190801	Pocillopora	16.8	5.95	4.05	✓
1343	20190814	Pocillopora	11.7	8.55	1.45	✓
1415	20190815	Pocillopora	36.4	2.75	7.25	✓
1416	20191009	Pocillopora	7.1	14.08	-4.08	✓
1418	20190720	Pocillopora	31.2	3.21	6.79	✓
1427	20190807	Pocillopora	29.7	3.37	6.63	✓
1445	20190826	Pocillopora	26.1	3.83	6.17	✓
1451	20190720	Pocillopora	39.5	2.53	7.47	✓
1459	20190722	Pocillopora	50	2.00	8.00	✓
1466	20190731	Pocillopora	32.8	3.05	6.95	✓
1468	20191121	Pocillopora	15.95	6.27	3.73	✓
1471	20190718	Pocillopora	41.2	2.43	7.57	✓
1486	20190720	Pocillopora	37.2	2.69	7.31	✓
1487	20190807	Pocillopora	36.1	2.77	7.23	✓
1536	20190725	Pocillopora	58.2	1.72	8.28	✓
1542	20190815	Pocillopora	15.35	6.51	3.49	✓
1559	20190725	Pocillopora	35.4	2.82	7.18	✓
1563	20190905	Pocillopora	24	4.17	5.83	✓
1571	20191121	Pocillopora	25.7	3.89	6.11	✓
1581	20190826	Pocillopora	37.2	2.69	7.31	✓
1582	20190926	Pocillopora	20.9	4.78	5.22	✓
1594	20190720	Pocillopora	16.1	6.21	3.79	✓
1595	20190808	Pocillopora	57.6	1.74	8.26	✓
1596	20191009	Pocillopora	13.1	7.63	2.37	✓
1617	20190801	Pocillopora	29.9	3.34	6.66	✓
1637	20190805	Pocillopora	49.6	2.02	7.98	✓
1641	20190720	Pocillopora	26.9	3.72	6.28	✓
1642	20190903	Pocillopora	37.3	2.68	7.32	✓
1647	20190926	Pocillopora	59.3	1.69	8.31	✓
1653	20190826	Pocillopora	31.9	3.13	6.87	✓
1676	20190718	Pocillopora	23.5	4.26	5.74	✓
1696	20190718	Pocillopora	23.9	4.18	5.82	✓
1701	20190826	Pocillopora	34.2	2.92	7.08	✓
1707	20190905	Pocillopora	35.4	2.82	7.18	✓
1709	20191121	Pocillopora	20	5.00	5.00	✓
1721	20190805	Pocillopora	19.35	5.17	4.83	✓
1728	20190806	Pocillopora	48.1	2.08	7.92	✓
1732	20190724	Pocillopora	42.6	2.35	7.65	✓
1744	20190807	Pocillopora	21.8	4.59	5.41	✓
1755	20190801	Pocillopora	12	8.33	1.67	✓
1757	20190731	Pocillopora	31.4	3.18	6.82	✓
1762	20191121	Pocillopora	18.85	5.31	4.69	✓
1765	20190725	Pocillopora	50.5	1.98	8.02	✓
1767	20190823	Pocillopora	52.1	1.92	8.08	✓
1775	20190718	Pocillopora	37.2	2.69	7.31	✓
1777	20190930	Pocillopora	39.2	2.55	7.45	✓
1820	20190815	Pocillopora	21.4	4.67	5.33	✓
2002	20190826	Pocillopora	26.5	3.77	6.23	✓
2005	20190718	Pocillopora	30.3	3.30	6.70	✓
2012	20190730	Pocillopora	36.1	2.77	7.23	✓
2026	20190718	Pocillopora	14.85	6.73	3.27	✓
2064	20190730	Pocillopora	29.5	3.39	6.61	✓
2072	20190814	Pocillopora	20.2	4.95	5.05	✓
2087	20190826	Pocillopora	35.3	2.83	7.17	✓
2185	20191009	Pocillopora	15.9	6.29	3.71	✓
2195	20191121	Pocillopora	30	3.33	6.67	✓
2197	20190729	Pocillopora	25.4	3.94	6.06	✓
2202	20190724	Pocillopora	50.8	1.97	8.03	✓
2210	20190826	Pocillopora	21.4	4.67	5.33	✓
2212	20190903	Pocillopora	19.3	5.18	4.82	✓
2300	20190731	Pocillopora	33.3	3.00	7.00	✓
2304	20190722	Pocillopora	12.75	7.84	2.16	✓
2305	20190724	Pocillopora	83	1.20	8.80	✓
2306	20190926	Pocillopora	53.8	1.86	8.14	✓
2357	20190718	Pocillopora	61.8	1.62	8.38	✓
2363	20190720	Pocillopora	40.2	2.49	7.51	✓
2409	20190808	Pocillopora	55.7	1.80	8.20	✓
2413	20190809	Pocillopora	36.9	2.71	7.29	✓
2414	20190807	Pocillopora	48	2.08	7.92	✓
2513	20190722	Pocillopora	12.55	7.97	2.03	✓
2527	20190805	Pocillopora	61.2	1.63	8.37	✓
2534	20191208	Pocillopora	48.7	2.05	7.95	✓
2550	20190724	Pocillopora	60.4	1.66	8.34	✓
2564	20190726	Pocillopora	57.5	1.74	8.26	✓
2668	20191001	Pocillopora	31.1	3.22	6.78	✓
2733	20191113	Pocillopora	32.8	3.05	6.95	✓
2743	20190724	Pocillopora	101	0.99	9.01	✓
2750	20190905	Pocillopora	45.8	2.18	7.82	✓
2861	20190730	Pocillopora	25.3	3.95	6.05	✓
2870	20190801	Pocillopora	36	2.78	7.22	✓
2873	20190730	Pocillopora	44.2	2.26	7.74	✓
2877	20190731	Pocillopora	59.1	1.69	8.31	✓
2878	20190807	Pocillopora	48.7	2.05	7.95	✓
2879	20190930	Pocillopora	33.5	2.99	7.01	✓
2977	20190815	Pocillopora	14.75	6.78	3.22	✓
2979	20190722	Pocillopora	32.2	3.11	6.89	✓
2993	20190725	Pocillopora	48.4	2.07	7.93	✓
2999	20190801	Pocillopora	44	2.27	7.73	✓
1169	20191113	Pocillopora	28.4	3.52	6.48	✓

Notes:

• 1755: 7.6 uL of DNA and 2.4 uL of water added instead of original value; double check at qubit step that this isn’t too diluted. In the end we only need a small amount of DNA so it might be okay. Otherwise I can re-do this sample at the end.
• All dilutions done on 20200914
• In gel look for 1459 to make sure it has DNA, the 2.00 uL DNA addition was checked off (probably just missed physically checking this off on my printed sheet); same for 1775 and 2409
• 1169 done on 20201102-3 because it was originally left off the list. All other dilutions made at one time.

Dilution Plates

PLATE 1:

	1	2	3	4	5	6	7	8	9	10	11	12
A	1254	1676	2979	1471	NA	1757	2861	1238	1536	1418	1141	1219
B	1103	2363	2304	1594	2999	2357	1765	NA	1184	2993	1205	2513
C	2870	1227	1159	1296	1451	2026	1732	1644	2202	1459	2300	2305
D	1168	2005	2873	1134	2064	1051	1617	2564	2550	1330	2877	1721
E	1486	1303	1696	1225	1775	1466	NA	1220	2012	1755	2195	1302
F	1468	2733	1329	1239	1762	1709	1571	1147	2534	1207	2527	2668
G	2087	1653	2414	1415	1820	1637	1445	2002	1138	1595	1041	1090
H	1744	1343	1767	2878	2977	1043	1582	1281	1701	2210	NA	1581

PLATE 2:

	1	2	3	4	5	6	7	8	9	10	11	12
A	1647	1563	1542	1059	2306	1487	1047	2212	2409	1050	2413	1707
B	2879	1777	2185	1427	2072	1728	2750	1416	1642	1596	1060	2197
C	2743	1559	1169
D
E
F
G
H

Notes:

• 1581 has higher volume of liquid when I looked at the dilution plate. Check in qubit step that is sample isn’t too diluted.
• Plates are labeled on the side and with a foil seal on top. Stored at -20C.

PCR Reactions

PCR Plate Outline

Samples are put in columns 1, 4, 7, and 10 so that triplicates can be made in the adjacent two columns using the multi-chanel pipette (ex: 1254 will be in B1, B2, and B3 during the PCR run). See the protocol for details on running in triplicates.

Plate 1 - PCR made and run on 20200915

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control			1536			2999			1227
B	1254			1418			2357			1159
C	1676			1141			1765			1296
D	2979			1219			1184			1451
E	1471			1103			2993			2026
F	1757			2363			1205			1732
G	2861			2304			2513			1641
H	1238			1594			2870			Neg Control

Plate 2 - PCR made and run on 20200915

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control			2064			1486			1755
B	1459			1051			1303			2195
C	2300			1617			1696			1302
D	2305			2564			1225			1468
E	1168			2550			1775			2733
F	2005			1330			1466			1329
G	2873			2877			1220			1239
H	1131			1721			2012			Neg control

Plate 3

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control			2087			1138			2977
B	1709			1653			1595			1043
C	1571			2414			1041			1582
D	1147			1415			1090			1281
E	2534			1820			1744			1701
F	1207			1637			1343			2210
G	2527			1445			1767			1581
H	2668			2002			2878			Neg control

Plate 4

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control			2409			2072			2743
B	1563			1050			1728			1559
C	1542			2413			2750			2202
D	1059			1707			1416			1762
E	2306			2879			1642			1647
F	1487			1777			1596			Neg control
G	1047			2185			1060				Neg control	1169
H	2212			1427			2197				Neg control	1755

Sanity check: 30 samples for Plate 1, 2, and 3 (90 total), 28 samples for Plate 4 = 118 total samples.

STRIP TUBE 1 - 20201028 1755; Neg control

STRIP TUBE 2 - 20201102 1169; Neg control

Notes:

• If I have a full dilution plate (like plate 1), I should have had the PCR plate be identical to the columns in the full dilution plate so that I can multi-chanel pipette
• 1755 re-done at later date. The 1755 that worked is in PCR plate 4 well H12.
• 1169 was done at a later date because it was originally left off of the list. Well G12.

PCR Runs 20200915 - Plates 1 and 2 run

Master mix created for half of the samples. 60 samples, 4 negative controls (blanks), and 1 for potential pipetting error.

Master Mix	uL	# of samples (60 + 4 neg controls + 5 for error)	total needed (uL)
Phusion PCR master mix	50	69	3450
UltraPure water	44	69	3036
10uM working stock FatP6.1 primer	1.3	69	89.70
10uM working stock RORF primer	1.3	69	89.70

Sanity check that calculations are correct.
total volume MM = 6665.4 / 97 uL per well = 68.71546392 (number should equal calculated sample number above)

20201020 - Plates 3 and 4 run

Master mix created for half of the samples. 60 samples, 4 negative controls (blanks), and 1 for potential pipetting error.

Master Mix	uL	# of samples (60 + 4 neg controls + 5 for error)	total needed (uL)
Phusion PCR master mix	50	69	3450
UltraPure water	44	69	3036
10uM working stock FatP6.1 primer	1.3	69	89.70
10uM working stock RORF primer	1.3	69	89.70

Sanity check that calculations are correct.
total volume MM = 6665.4 / 97 uL per well = 68.71546392 (number should equal calculated sample number above)

20201028 - 1 sample and 1 control in a strip tube

Master Mix	uL	# of samples (1 + 1 control + 0.1 error)	total needed (uL)
Phusion PCR master mix	50	2.1	105
UltraPure water	44	2.1	92.4
10uM working stock FatP6.1 primer	1.3	2.1	2.73
10uM working stock RORF primer	1.3	2.1	2.73

Sanity check that calculations are correct. total volume MM = 202.86
/ 97 uL per well = 2.09 (should be equal to sample number above)

20201102 - 1 sample and 1 control in a strip tube

Master Mix	uL	# of samples (1 + 1 control + 0.1 error)	total needed (uL)
Phusion PCR master mix	50	2.1	105
UltraPure water	44	2.1	92.4
10uM working stock FatP6.1 primer	1.3	2.1	2.73
10uM working stock RORF primer	1.3	2.1	2.73

Sanity check that calculations are correct. total volume MM = 202.86
/ 97 uL per well = 2.09 (should be equal to sample number above)

1X Bead Clean-Up

20200915 Plates 1 and 2 cleaned. Stored in -20C freezer waiting to quantified.
No notes necessary, protocol followed exactly (see link at the top of this post).
End result is 50 uL of sample in each well that look identical to the PCR platemaps above.

20201025 Plates 3 and 4 cleaned. No notes; see above.

20201028: 1755 bead clean-up: No notes. 20201103 1169 bead clean up: No notes.

All stored in -20C freezer when not in use.

Qubit (ng/uL) Values

20200922: BR DNA Standard 1: 172.65 Standard 2: 16,875.12.

• Original standard 2 was 12,416.92 but re-made because thought that was much lower than normal.

20201026: BR DNA Standard 1: 200.79 Standard 2: 22,255.60.
20201028: BR DNA Standard 1: 184.39 Standard 2: 20,275.06.
20201103: BR DNA Standard 1: 218.44 Standard 2: 24,795.11.

Qubit Date	Sample ID	DNA 1	DNA 2	Average DNA	Gel Date	Gel Pass?
20200922	1594	6.7	6.76	6.73	2020923	Yes
20200922	1205	37.2	36.8	37	2020923	Yes
20200922	2513	26.4	26.2	26.3	2020923	Yes
20200922	2005	11.9	11.8	11.85	2020923	Yes
20200922	1732	20	19.9	19.95	2020923	Yes
20200922	1721	15.2	15.1	15.15	2020923	Yes
20200922	2733	19.7	19.5	19.6	2020923	Yes
20200922	1254	12.9	12.8	12.85	20201026	Yes
20200922	1676	19.4	19.2	19.3	20201026	Yes
20201026	2979	18.1	17.9	18	20201026	Yes
20200922	1471	19.2	19	19.1	20201026	Yes
20200922	1757	17.3	17.2	17.25	20201026	Yes
20200922	2861	20.2	20	20.1	20201026	Yes
20200922	1238	19.8	19.7	19.75	20201026	Yes
20200922	1536	9.46	9.32	9.39	20201026	Yes
20200922	1418	8.8	8.74	8.77	20201026	Yes
20200922	1141	8.74	8.66	8.7	20201026	Yes
20200922	1219	10.6	10.6	10.6	20201026	Yes
20200922	1103	8.46	8.38	8.42	20201026	Yes
20200922	2304	5.3	5.22	5.26	20201026	Yes
20200922	2999	23.8	23.6	23.7	20201026	Yes
20200922	2357	24.8	24.6	24.7	20201026	Yes
20200922	1765	26	25.8	25.9	20201026	Yes
20200922	1184	29.8	29.6	29.7	20201026	Yes
20200922	2993	34	33.6	33.8	20201026	Yes
20200922	2366	8.8	8.52	8.66	20201026	Yes
20200922	1159	31.8	31.4	31.6	20201026	Yes
20200922	1296	31.2	31	31.1	20201026	Yes
20200922	1451	34.8	34.4	34.6	20201026	Yes
20200922	1227	32	31.6	31.8	20201026	Yes
20200922	2026	32.2	31.8	32	20201026	Yes
20200922	2870	24.6	24.4	24.5	20201026	Yes
20200922	1459	14.8	14.6	14.7	20201026	Yes
20200922	2873	16.9	16.5	16.7	20201026	Yes
20200922	2300	20	19.8	19.9	20201026	Yes
20200922	1641	27.4	27.2	27.3	20201026	Yes
20200922	2305	15.1	15	15.05	20201026	Yes
20200922	1168	15.7	15.5	15.6	20201026	Yes
20200922	1131	17.2	17	17.1	20201026	Yes
20200922	2877	19.7	19.5	19.6	20201026	Yes
20200922	1617	30	29.8	29.9	20201026	Yes
20200922	2550	28.6	28.4	28.5	20201026	Yes
20200922	2064	32.4	32.2	32.3	20201026	Yes
20200922	1330	13.7	13.6	13.65	20201026	Yes
20200922	1051	26.6	26.4	26.5	20201026	Yes
20200922	2564	22.4	21.8	22.1	20201026	Yes
20200922	1696	14.5	14.2	14.35	20201026	Yes
20201026	1225	16.6	16.1	16.35	20201026	Yes
20200922	1486	20.8	20.6	20.7	20201026	Yes
20200922	1220	3.48	3.42	3.45	20201026	Yes
20200922	1466	12.2	12	12.1	20201026	Yes
20200922	1303	14.1	13.8	13.95	20201026	Yes
20200922	1775	15.2	15.1	15.15	20201026	Yes
20200922	1302	27.2	27	27.1	20201026	Yes
20200922	1329	24.2	24	24.1	20201026	Yes
20200922	1468	26	25.8	25.9	20201026	Yes
20201028	1755	7.2	7.14	7.17	20201028	Yes
20200922	1239	23.2	23	23.1	20201026	Yes
20200922	2012	22.8	22.6	22.7	20201026	Yes
20200922	2195	20.8	20.6	20.7	20201026	Yes
20201026	2534	15.9	15.4	15.65	20201026	Yes
20201026	1709	13.5	13.1	13.3	20201026	Yes
20201026	1571	12.1	11.7	11.9	20201026	Yes
20201026	2527	17.5	16.8	17.15	20201026	Yes
20201026	1147	14.9	14.5	14.7	20201026	Yes
20201026	2668	17.5	16.9	17.2	20201026	Yes
20201026	1207	17	16.4	16.7	20201026	Yes
20201026	1445	7.26	6.96	7.11	20201026	Yes
20201026	2414	6.86	6.58	6.72	20201026	Yes
20201026	2002	7.7	7.36	7.53	20201026	Yes
20201026	1820	4.84	4.66	4.75	20201026	Yes
20201026	1637	6.44	6.08	6.26	20201026	Yes
20201026	2087	5.68	5.48	5.58	20201026	Yes
20201026	1415	5.4	5.12	5.26	20201026	Yes
20201026	1653	7.58	7.3	7.44	20201026	Yes
20201026	1041	10.2	9.84	10.02	20201026	Yes
20201026	1595	12.8	12.6	12.7	20201026	Yes
20201026	1138	6.08	5.94	6.01	20201026	Yes
20201026	1090	11.6	11.3	11.45	20201026	Yes
20201026	1744	13.5	13.3	13.4	20201026	Yes
20201026	1343	8.92	8.68	8.8	20201026	Yes
20201026	1767	14.6	14.2	14.4	20201026	Yes
20201026	2878	15.1	14.8	14.95	20201026	Yes
20201026	1582	19.9	19.5	19.7	20201026	Yes
20201026	1045	19.9	19.1	19.5	20201026	Yes
20201026	2977	17.9	17.4	17.65	20201026	Yes
20201026	1281	23.4	22.8	23.1	20201026	Yes
20201026	1701	26.2	25.8	26	20201026	Yes
20201026	2210	23.2	22.6	22.9	20201026	Yes
20201026	1581	24.6	24.2	24.4	20201026	Yes
20201026	1060	19.2	18.4	18.8	20201026	Yes
20201026	2197	18	17.4	17.7	20201026	Yes
20201026	2202	8.64	8.22	8.43	20201026	Yes
20201026	2743	8.92	8.6	8.76	20201026	Yes
20201026	1762	10.9	10.6	10.75	20201026	Yes
20201026	1559	8.84	8.38	8.61	20201026	Yes
20201026	1487	19.7	19	19.35	20201026	Yes
20201026	1047	22.4	21.4	21.9	20201026	Yes
20201026	2409	12.7	12.2	12.45	20201026	Yes
20201026	1647	10.5	10.3	10.4	20201026	Yes
20201026	2212	19.7	19	19.35	20201026	Yes
20201026	2750	16.2	15.7	15.95	20201026	Yes
20201026	2185	12.2	11.1	11.65	20201026	Yes
20201026	1059	22.8	22	22.4	20201026	Yes
20201026	2072	15.7	15.3	15.5	20201026	Yes
20201026	1563	19.4	18.5	18.95	20201026	Yes
20201026	1642	15.6	15.1	15.35	20201026	Yes
20201026	1596	19	18.4	18.7	20201026	Yes
20201026	1050	13.2	12.9	13.05	20201026	Yes
20201026	1728	16.2	15.3	15.75	20201026	Yes
20201026	1777	12.5	12.2	12.35	20201026	Yes
20201026	1427	13.9	13.5	13.7	20201026	Yes
20201026	1416	16.3	15.7	16	20201026	Yes
20201026	2879	13.3	13	13.15	20201026	Yes
20201026	1707	13.6	13.3	13.45	20201026	Yes
20201103	1169	15.6	15.5	15.55	20201103	Yes

Those with ** for values were too low to measure. I will re-qubit these in case 1 full uL wasn’t added to the qubit tube. Otherwise there might not have been a full uL added to the PCR plate.

Gel Electrophoresis Analysis

mtORF amplication will result in a sharp band ~1,000 bp.

20200923 Gel:

20201026 Gels:

20201028 Gel:

20201103 Gel:

Sequencing Prep

20200923: A small set of 7 samples (HPE01-07) were chosen to analyze on a gel before sending to sequence as a test run. These samples were chosen randomly but I tried to make sure a range of qubit values were included (6 - 30 ng) and samples from both PCR plates were included from various columns.

20201103: The rest of the samples were dropped off for sequencing (HPE08-119).

1:2 dilution chosen; 5 uL of DNA and 5 uL of water added.

Prep Date	Sample ID	DNA 1	DNA 2	Average DNA(ng_uL)	1:2 Dilutions (ng_uL)	DNA to 25 ng	H2O to 10 uL	Notes
20200924	1205	37.2	36.8	37	18.50	1.35	8.65	✓
20200924	1594	6.7	6.76	6.73	3.37	7.43	2.57	✓
20200924	1721	15.2	15.1	15.15	7.58	3.30	6.70	✓
20200924	1732	20	19.9	19.95	9.98	2.51	7.49	✓
20200924	2005	11.9	11.8	11.85	5.93	4.22	5.78	✓
20200924	2513	26.4	26.2	26.3	13.15	1.90	8.10	✓
20200924	2733	19.7	19.5	19.6	9.80	2.55	7.45	✓
20201102	1169	15.5	15.6	15.55	7.78	3.22	6.78	✓
20201028-9	1041	10.2	9.84	10.02	5.01	4.99	5.01	✓
20201028-9	1043	26	26	26	13.00	1.92	8.08	✓
20201028-9	1047	22.4	21.4	21.9	10.95	2.28	7.72	✓
20201028-9	1050	13.2	12.9	13.05	6.53	3.83	6.17	✓
20201028-9	1051	26.6	26.4	26.5	13.25	1.89	8.11	✓
20201028-9	1059	22.8	22	22.4	11.20	2.23	7.77	✓
20201028-9	1060	19.2	18.4	18.8	9.40	2.66	7.34	✓
20201028-9	1090	11.6	11.3	11.45	5.73	4.37	5.63	✓
20201028-9	1103	8.46	8.38	8.42	4.21	5.94	4.06	✓
20201028-9	1131	17.2	17	17.1	8.55	2.92	7.08	✓
20201028-9	1138	6.08	5.94	6.01	3.01	8.32	1.68	✓
20201028-9	1141	8.74	8.66	8.7	4.35	5.75	4.25	✓
20201028-9	1147	14.9	14.5	14.7	7.35	3.40	6.60	✓
20201028-9	1159	31.8	31.4	31.6	15.80	1.58	8.42	✓
20201028-9	1168	15.7	15.5	15.6	7.80	3.21	6.79	✓
20201028-9	1184	29.8	29.6	29.7	14.85	1.68	8.32	✓
20201028-9	1207	17	16.4	16.7	8.35	2.99	7.01	✓
20201028-9	1219	10.6	10.6	10.6	5.30	4.72	5.28	✓
20201028-9	1220	3.48	3.42	3.45	3.435	7.25	2.75	Not diluted; ✓
20201028-9	1225	16.6	16.1	16.35	8.18	3.06	6.94	✓
20201028-9	1227	32	31.6	31.8	15.90	1.57	8.43	✓
20201028-9	1238	19.8	19.7	19.75	9.88	2.53	7.47	✓
20201028-9	1239	23.2	23	23.1	11.55	2.16	7.84	✓
20201028-9	1254	12.9	12.8	12.85	6.43	3.89	6.11	✓
20201028-9	1281	23.4	22.8	23.1	11.55	2.16	7.84	✓
20201028-9	1296	31.2	31	31.1	15.55	1.61	8.39	✓
20201028-9	1302	27.2	27	27.1	13.55	1.85	8.15	✓
20201028-9	1303	14.1	13.8	13.95	6.98	3.58	6.42	✓
20201028-9	1329	24.2	24	24.1	12.05	2.07	7.93	✓
20201028-9	1330	13.7	13.6	13.65	6.83	3.66	6.34	✓
20201028-9	1343	8.92	8.68	8.8	4.40	5.68	4.32	✓
20201028-9	1415	5.4	5.12	5.26	2.63	9.51	0.49	✓
20201028-9	1416	16.3	15.7	16	8.00	3.13	6.88	✓
20201028-9	1418	8.8	8.74	8.77	4.39	5.70	4.30	✓
20201028-9	1427	13.9	13.5	13.7	6.85	3.65	6.35	✓
20201028-9	1445	7.26	6.96	7.11	3.56	7.03	2.97	✓
20201028-9	1451	34.8	34.4	34.6	17.30	1.45	8.55	✓
20201028-9	1459	14.8	14.6	14.7	7.35	3.40	6.60	✓
20201028-9	1466	12.2	12	12.1	6.05	4.13	5.87	✓
20201028-9	1468	26	25.8	25.9	12.95	1.93	8.07	✓
20201028-9	1471	19.2	19	19.1	9.55	2.62	7.38	✓
20201028-9	1486	20.8	20.6	20.7	10.35	2.42	7.58	✓
20201028-9	1487	19.7	19	19.35	9.68	2.58	7.42	✓
20201028-9	1536	9.46	9.32	9.39	4.70	5.32	4.68	✓
20201028-9	1542	19.4	19.4	19.4	9.70	2.58	7.42	✓
20201028-9	1559	8.84	8.38	8.61	4.31	5.81	4.19	✓
20201028-9	1563	19.4	18.5	18.95	9.48	2.64	7.36	✓
20201028-9	1571	12.1	11.7	11.9	5.95	4.20	5.80	✓
20201028-9	1581	24.6	24.2	24.4	12.20	2.05	7.95	✓
20201028-9	1582	19.9	19.5	19.7	9.85	2.54	7.46	✓
20201028-9	1595	12.8	12.6	12.7	6.35	3.94	6.06	✓
20201028-9	1596	19	18.4	18.7	9.35	2.67	7.33	✓
20201028-9	1617	30	29.8	29.9	14.95	1.67	8.33	✓
20201028-9	1637	6.44	6.08	6.26	3.13	7.99	2.01	✓
20201028-9	1641	27.4	27.2	27.3	13.65	1.83	8.17	✓
20201028-9	1642	15.6	15.1	15.35	7.68	3.26	6.74	✓
20201028-9	1647	10.5	10.3	10.4	5.20	4.81	5.19	✓
20201028-9	1653	7.58	7.3	7.44	3.72	6.72	3.28	✓
20201028-9	1676	19.4	19.2	19.3	9.65	2.59	7.41	✓
20201028-9	1696	14.5	14.2	14.35	7.18	3.48	6.52	✓
20201028-9	1701	26.2	25.8	26	13.00	1.92	8.08	✓
20201028-9	1707	13.6	13.3	13.45	6.73	3.72	6.28	✓
20201028-9	1709	13.5	13.1	13.3	6.65	3.76	6.24	✓
20201028-9	1728	16.2	15.3	15.75	7.88	3.17	6.83	✓
20201028-9	1744	13.5	13.3	13.4	6.70	3.73	6.27	✓
20201028-9	1755	7.2	7.14	7.17	3.59	6.97	3.03	✓
20201028-9	1757	17.3	17.2	17.25	8.63	2.90	7.10	✓
20201028-9	1762	10.9	10.6	10.75	5.38	4.65	5.35	✓
20201028-9	1765	26	25.8	25.9	12.95	1.93	8.07	✓
20201028-9	1767	14.6	14.2	14.4	7.20	3.47	6.53	✓
20201028-9	1775	15.2	15.1	15.15	7.58	3.30	6.70	✓
20201028-9	1777	12.5	12.2	12.35	6.18	4.05	5.95	✓
20201028-9	1820	4.84	4.66	4.75	4.705	5.26	4.74	Not diluted; ✓
20201028-9	2002	7.7	7.36	7.53	3.77	6.64	3.36	✓
20201028-9	2012	22.8	22.6	22.7	11.35	2.20	7.80	✓
20201028-9	2026	32.2	31.8	32	16.00	1.56	8.44	✓
20201028-9	2064	32.4	32.2	32.3	16.15	1.55	8.45	✓
20201028-9	2072	15.7	15.3	15.5	7.75	3.23	6.77	✓
20201028-9	2087	5.68	5.48	5.58	2.79	8.96	1.04	✓
20201028-9	2185	12.2	11.1	11.65	5.83	4.29	5.71	✓
20201028-9	2195	20.8	20.6	20.7	10.35	2.42	7.58	✓
20201028-9	2197	18	17.4	17.7	8.85	2.82	7.18	✓
20201028-9	2202	8.64	8.22	8.43	4.22	5.93	4.07	✓
20201028-9	2210	23.2	22.6	22.9	11.45	2.18	7.82	✓
20201028-9	2212	19.7	19	19.35	9.68	2.58	7.42	✓
20201028-9	2300	20	19.8	19.9	9.95	2.51	7.49	✓
20201028-9	2304	5.3	5.22	5.26	2.63	9.51	0.49	✓
20201028-9	2305	15.1	15	15.05	7.53	3.32	6.68	✓
20201028-9	2306	26.2	26	26.1	13.05	1.92	8.08	✓
20201028-9	2357	24.8	24.6	24.7	12.35	2.02	7.98	✓
20201028-9	2363	5.24	5.2	5.22	2.61	9.58	0.42	✓
20201028-9	2409	12.7	12.2	12.45	6.23	4.02	5.98	✓
20201028-9	2413	13.7	13.7	13.7	6.85	3.65	6.35	✓
20201028-9	2414	6.86	6.58	6.72	3.36	7.44	2.56	✓
20201028-9	2527	17.5	16.8	17.15	8.58	2.92	7.08	✓
20201028-9	2534	15.9	15.4	15.65	7.83	3.19	6.81	✓
20201028-9	2550	28.6	28.4	28.5	14.25	1.75	8.25	✓
20201028-9	2564	22.4	21.8	22.1	11.05	2.26	7.74	✓
20201028-9	2668	17.5	16.9	17.2	8.60	2.91	7.09	✓
20201028-9	2743	8.92	8.6	8.76	4.38	5.71	4.29	✓
20201028-9	2750	16.2	15.7	15.95	7.98	3.13	6.87	✓
20201028-9	2861	20.2	20	20.1	10.05	2.49	7.51	✓
20201028-9	2870	24.6	24.4	24.5	12.25	2.04	7.96	✓
20201028-9	2873	16.9	16.5	16.7	8.35	2.99	7.01	✓
20201028-9	2877	19.7	19.5	19.6	9.80	2.55	7.45	✓
20201028-9	2878	15.1	14.8	14.95	7.48	3.34	6.66	✓
20201028-9	2879	13.3	13	13.15	6.58	3.80	6.20	✓
20201028-9	2977	17.9	17.4	17.65	8.83	2.83	7.17	✓
20201028-9	2979	18.1	17.9	18	9.00	2.78	7.22	✓
20201028-9	2993	34	33.6	33.8	16.90	1.48	8.52	✓
20201028-9	2999	23.8	23.6	23.7	11.85	2.11	7.89	✓
20201028-9	NA_1045	19.9	19.1	19.5	9.75	2.56	7.44	Not on list? Qubit # miswritten
20201028-9	NA_2366	8.8	8.52	8.66	4.33	5.77	4.23	Not on list? Qubit # miswritten

See the Putnam lab mtORF protocol for the URI GSC sequencing forms.

1:2 Dilution platemaps

Plate 1

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control	1536	2999	1227	Neg Control	2064	1486	1755	Neg Control	2087	1138	2977
B	1254	1418	2357	1159	1459	1051	1303	2195	1709	1653	1595	1043
C	1676	1141	1765	1296	2300	1617	1696	1302	1571	2414	1041	1582
D	2979	1219	1184	1451	2305	2564	1225	1468	1147	1415	1090	1281
E	1471	1103	2993	2026	1168	2550	1775	2733	2534	1820	1744	1701
F	1757	2363	1205	1732	2005	1330	1466	1329	1207	1637	1343	2210
G	2861	2304	2513	1641	2873	2877	1220	1239	2527	1445	1767	1581
H	1238	1594	2870	Neg Control	1131	1721	2012	Neg control	2668	2002	2878	Neg control

Plate 2

	1	2	3	4	5	6	7	8	9	10	11	12
A	Neg Control	2409	2072	2743
B	1563	1050	1728	1559
C	1542	2413	2750	2202
D	1059	1707	1416	1762
E	2306	2879	1642	1647
F	1487	1777	1596	Neg control
G	1047	2185	1060	1755
H	2212	1427	2197	1169

25 ng platemaps that were dropped off for sequencing

10 uL of 25ng DNA sample and 2 uL of 3.2 uM primer is in each well.

Plate 1

	1	2	3	4	5	6	7	8	9	10	11	12
A	1254	1676	2179	1471	1757	2861	1238	1536	1418	1141	1219	1103
B	2304	2999	2357	1765	1184	2993	2870	1227	1159	1296	1451	2026
C	1641	1459	2300	2305	1168	2873	1131	2064	1051	1617	2564	2550
D	1330	2877	1486	1303	1696	1225	1775	1466	1220	2012	2195	1302
E	1468	1329	1239	1709	1571	1147	2534	1207	2527	2668	2087	1653
F	2414	1415	1820	1637	1445	2002	1138	1595	1041	1090	1744	1343
G	1767	2878	2977	1582	1281	1701	2210	1581	1563	1059	1487	1047
H	2212	2409	1050	1707	2879	1777	2185	1427	2072	1728	2750	1416

Plate 2

	1	2	3	4	5	6	7	8	9	10	11	12
A	1642	2743
B	1542	1559
C	1596	2202
D	2306	1762
E	1060	1647
F	2413	1755
G	2197	2363
H	1169	1043

3.2 uM calculations

20201028:
3.2 uM primer = 68.48 uL 10 uM primer + 145.52 uL ultrapure water

20201103: x = (3.2 uM Fat6.1 primer)(3 uL total)/10 uM Fat6.1 primer = 0.96 uL 10 uM Fat6.1 primer
3.2 uM primer = 0.96 uL 10 uM primer + 2.04 ultrapure water

2 samples did not run in sequencing

I uploaded sequences to the program Geneious to align all 119 sequences returned from GSC and determine which 2 didn’t work. HPE70 and HPE78 sequences were the two files with no sequences, which were 1581 and 1563.

My initial thought was that the primer wasn’t added correctly to the 25ng dilution plates that were given to GSC. So, I re-qubited the samples from the 1:2 dilution plates to make sure there was DNA added. Below are those results.

BR DNA Standard 1: 193.10
BR DNA Standard 2: 22,836.21

1581 DNA 1: 11.8, DNA 2: 11.7, Average: 11.75 ng/uL.
1563 DNA 1: 8.42, DNA 2: 8.32, Average: 8.37 ng/uL.

From this, it was most likely an issue with adding primer or DNA to the 25 ng plate.

25 ng plate calculations:

1581 (11.75 ng/uL): 2.13 uL DNA + 7.87 uL Ultrapure H2O + 2 uL 3.2 uM primer
1563 (8.32 ng/uL): 2.99 uL DNA + 7.01 uL Ultrapure H2O + 2 uL 3.2 uM primer

4 uL of 3.2 uM primer = 1.28 uL 10 uM primer + 2.72 uL Ultrapure H2O

HPE120 = 1581
HPE121 = 1563

These samples were re-made in PCR strip tubes labeled HPE120 and HPE121, then placed in Janet/GSC’s freezer on 20201111.