Jake Heare Research Central

Thursday, May 7, 2015

5 7 2015 Oly Seed DNA check for GBS

Today I checked all the seed DNA isolations using the nanodrop to get concentrations and quality checks done for the Oly GBS project. You can read the results below.

Sample ID	User ID	Date	Time	ng/ul	A260	A280	260/280	260/230	Constant	Cursor Pos.	Cursor abs.	340 raw
HL1	Default	5/7/2015	10:16 AM	39.16	0.979	0.515	1.9	1.7	40	230	0.576	0.016
HL2	Default	5/7/2015	10:17 AM	141.47	3.537	1.749	2.02	2.27	40	230	1.559	0.043
HL3	Default	5/7/2015	10:18 AM	58.05	1.451	0.716	2.03	2.19	40	230	0.664	0.024
HL4	Default	5/7/2015	10:19 AM	119.39	2.985	1.504	1.98	2.27	40	230	1.314	0.064
HL5	Default	5/7/2015	10:20 AM	64.32	1.608	0.805	2	2.2	40	230	0.732	0.053
HL6	Default	5/7/2015	10:21 AM	78.93	1.973	0.976	2.02	2.22	40	230	0.89	0.033
HL7	Default	5/7/2015	10:22 AM	115.35	2.884	1.419	2.03	2.3	40	230	1.251	0.049
HL8	Default	5/7/2015	10:23 AM	11.77	0.235	0.129	1.82	1.54	50	230	0.153	0.055
HL9	Default	5/7/2015	10:25 AM	172.45	3.449	1.726	2	2.31	50	230	1.495	0.043
HL10	Default	5/7/2015	10:26 AM	96.08	1.922	0.966	1.99	2.2	50	230	0.875	0.029
HL11	Default	5/7/2015	10:27 AM	192.3	3.846	1.951	1.97	2.28	50	230	1.689	0.081
HL12	Default	5/7/2015	10:28 AM	58.12	1.162	0.584	1.99	2.15	50	230	0.542	0.047
HL13	Default	5/7/2015	10:29 AM	43.74	0.875	0.451	1.94	2.08	50	230	0.421	0.015
HL14	Default	5/7/2015	10:30 AM	127.92	2.558	1.275	2.01	2.35	50	230	1.09	0.03
HL15	Default	5/7/2015	10:31 AM	28.71	0.574	0.305	1.88	2.01	50	230	0.285	0.015
HL16	Default	5/7/2015	10:32 AM	98.46	1.969	0.978	2.01	2.3	50	230	0.858	0.012
HL17	Default	5/7/2015	10:33 AM	73.78	1.476	0.724	2.04	2.32	50	230	0.635	0.037
HL18	Default	5/7/2015	10:34 AM	66.94	1.339	0.668	2	2.22	50	230	0.604	0.044
HL19	Default	5/7/2015	10:35 AM	287.52	5.75	2.813	2.04	2.34	50	230	2.453	0.019
HL20	Default	5/7/2015	10:36 AM	144.94	2.899	1.412	2.05	2.33	50	230	1.246	0.038
HL21	Default	5/7/2015	10:37 AM	356.18	7.124	3.63	1.96	2.35	50	230	3.026	0.04
HL22	Default	5/7/2015	10:38 AM	222.98	4.46	2.234	2	2.39	50	230	1.869	0.019
HL23	Default	5/7/2015	10:39 AM	393.05	7.861	4.097	1.92	2.33	50	230	3.371	0.035
HL24	Default	5/7/2015	10:40 AM	60.32	1.206	0.62	1.95	2.11	50	230	0.571	0.076
HL25	Default	5/7/2015	10:41 AM	152.2	3.044	1.549	1.96	2.3	50	230	1.321	0.217
HL25	Default	5/7/2015	10:42 AM	142.1	2.842	1.41	2.02	2.67	50	230	1.066	-0.304
HL25	Default	5/7/2015	10:43 AM	128.42	2.568	1.29	1.99	2.29	50	230	1.122	0.097
HL26	Default	5/7/2015	10:44 AM	336.54	6.731	3.345	2.01	2.41	50	230	2.797	-0.006
HL27	Default	5/7/2015	10:45 AM	377.31	7.546	3.813	1.98	2.32	50	230	3.251	0.086
HL28	Default	5/7/2015	10:46 AM	103.32	2.066	1.07	1.93	2.24	50	230	0.921	0.085
HL29	Default	5/7/2015	10:47 AM	74.49	1.49	0.768	1.94	2.2	50	230	0.676	0.047
HL30	Default	5/7/2015	10:48 AM	274.08	5.482	2.804	1.95	2.26	50	230	2.427	0.207
HL31	Default	5/7/2015	10:50 AM	94.38	1.888	0.947	1.99	2.18	50	230	0.866	0.293
HL32	Default	5/7/2015	10:51 AM	269.6	5.392	2.786	1.94	2.16	50	230	2.499	0.3
NF1	Default	5/7/2015	10:53 AM	330.81	6.616	3.396	1.95	2.32	50	230	2.851	0.025
NF1	Default	5/7/2015	10:54 AM	356.37	7.127	3.676	1.94	2.31	50	230	3.09	0.044
NF2	Default	5/7/2015	10:55 AM	210.64	4.213	2.199	1.92	2.35	50	230	1.79	0.043
NF3	Default	5/7/2015	10:56 AM	242.37	4.847	2.514	1.93	2.21	50	230	2.196	0.254
NF4	Default	5/7/2015	10:57 AM	207.32	4.146	2.201	1.88	2	50	230	2.077	2.737
NF5	Default	5/7/2015	10:58 AM	323.45	6.469	3.368	1.92	2.11	50	230	3.066	0.363
NF6	Default	5/7/2015	10:59 AM	317.97	6.359	3.327	1.91	2.35	50	230	2.711	0.065
NF6	Default	5/7/2015	11:00 AM	270.17	5.403	2.794	1.93	2.38	50	230	2.271	0.077
NF7	Default	5/7/2015	11:01 AM	191.55	3.831	1.985	1.93	2.27	50	230	1.686	0.07
NF8	Default	5/7/2015	11:02 AM	361.84	7.237	3.677	1.97	2.33	50	230	3.102	0.04
NF9	Default	5/7/2015	11:03 AM	166.86	3.337	1.677	1.99	2.31	50	230	1.447	0.074
NF10	Default	5/7/2015	11:05 AM	124.73	2.495	1.255	1.99	2.22	50	230	1.125	0.135
NF11	Default	5/7/2015	11:06 AM	447.26	8.945	4.552	1.97	2.25	50	230	3.982	0.394
NF12	Default	5/7/2015	11:07 AM	93.36	1.867	0.963	1.94	2.08	50	230	0.896	0.138
NF13	Default	5/7/2015	11:08 AM	277	5.54	2.847	1.95	2.18	50	230	2.547	0.231
NF14	Default	5/7/2015	11:09 AM	298.5	5.97	2.983	2	2.32	50	230	2.576	0.053
NF15	Default	5/7/2015	11:10 AM	155.67	3.113	1.569	1.98	2.3	50	230	1.352	0.051
NF16	Default	5/7/2015	11:11 AM	135.04	2.701	1.396	1.94	2.15	50	230	1.258	0.139
NF17	Default	5/7/2015	11:13 AM	52.7	1.054	0.559	1.88	2.14	50	230	0.492	0.038
NF18	Default	5/7/2015	11:14 AM	292.28	5.846	2.973	1.97	2.21	50	230	2.646	0.311
NF19	Default	5/7/2015	11:15 AM	90.2	1.804	0.934	1.93	2.25	50	230	0.801	0.049
NF20	Default	5/7/2015	11:16 AM	374.23	7.485	3.772	1.98	2.36	50	230	3.176	0.034
NF20	Default	5/7/2015	11:17 AM	876.31	17.526	8.649	2.03	2.24	50	230	7.826	0.316
NF21	Default	5/7/2015	11:18 AM	233.53	4.671	2.416	1.93	2.37	50	230	1.971	0.057
NF22	Default	5/7/2015	11:19 AM	86.59	1.732	0.893	1.94	2.35	50	230	0.736	0.018
NF23	Default	5/7/2015	11:20 AM	204.91	4.098	2.067	1.98	2.35	50	230	1.741	0.034
NF24	Default	5/7/2015	11:21 AM	231.38	4.628	2.391	1.94	2.34	50	230	1.981	0.069
NF25	Default	5/7/2015	11:22 AM	126.3	2.526	1.384	1.82	1.59	50	230	1.593	0.863
NF26	Default	5/7/2015	11:23 AM	159.12	3.182	1.597	1.99	2.31	50	230	1.378	0.103
NF27	Default	5/7/2015	11:25 AM	101.21	2.024	1.058	1.91	2.33	50	230	0.869	0.035
NF28	Default	5/7/2015	11:26 AM	128.03	2.561	1.279	2	2.33	50	230	1.101	0.046
NF29	Default	5/7/2015	11:27 AM	176.44	3.529	1.786	1.98	2.33	50	230	1.515	0.038
NF30	Default	5/7/2015	11:28 AM	187.06	3.741	1.895	1.97	2.31	50	230	1.62	0.061
NF31	Default	5/7/2015	11:29 AM	117.53	2.351	1.179	1.99	2.27	50	230	1.036	0.082
NF32	Default	5/7/2015	11:30 AM	272.95	5.459	2.808	1.94	2.28	50	230	2.394	-0.407
SN1	Default	5/7/2015	11:36 AM	107.82	2.156	1.096	1.97	2.16	50	230	0.998	0.099
SN2	Default	5/7/2015	11:37 AM	278.11	5.562	2.803	1.98	2.26	50	230	2.464	0.124
SN3	Default	5/7/2015	11:38 AM	122.94	2.459	1.255	1.96	2.27	50	230	1.083	0.028
SN4	Default	5/7/2015	11:39 AM	225.29	4.506	2.236	2.01	2.31	50	230	1.949	0.706
SN5	Default	5/7/2015	11:41 AM	89.6	1.792	0.914	1.96	2.21	50	230	0.811	0.056
SN6	Default	5/7/2015	11:42 AM	79.5	1.59	0.808	1.97	2.23	50	230	0.712	0.044
SN7	Default	5/7/2015	11:43 AM	107.8	2.156	1.117	1.93	2.2	50	230	0.978	0.086
SN8	Default	5/7/2015	11:44 AM	212.99	4.26	2.185	1.95	2.25	50	230	1.89	0.417
SN9	Default	5/7/2015	11:45 AM	115.94	2.319	1.206	1.92	2.29	50	230	1.01	0.094
SN10	Default	5/7/2015	11:46 AM	118.82	2.376	1.221	1.95	2.21	50	230	1.074	0.085
SN11	Default	5/7/2015	11:47 AM	267.08	5.342	2.724	1.96	2.32	50	230	2.298	0.105
SN12	Default	5/7/2015	11:49 AM	127.94	2.559	1.317	1.94	2.32	50	230	1.103	0.071
SN13	Default	5/7/2015	11:50 AM	317.61	6.352	3.194	1.99	2.32	50	230	2.74	0.11
SN14	Default	5/7/2015	11:51 AM	146.97	2.939	1.498	1.96	2.23	50	230	1.316	0.08
SN15	Default	5/7/2015	11:52 AM	164.33	3.287	1.665	1.97	2.24	50	230	1.469	0.099
SN16	Default	5/7/2015	11:53 AM	77.75	1.555	0.821	1.89	1.87	50	230	0.833	0.312
SN17	Default	5/7/2015	11:54 AM	125.5	2.51	1.293	1.94	2.24	50	230	1.119	0.025
SN18	Default	5/7/2015	11:56 AM	170.94	3.419	1.748	1.96	2.35	50	230	1.456	0.023
SN19	Default	5/7/2015	11:58 AM	74.9	1.498	0.786	1.91	2.07	50	230	0.724	0.054
SN20	Default	5/7/2015	11:59 AM	104.44	2.089	1.078	1.94	2.33	50	230	0.895	0.026
SN21	Default	5/7/2015	12:00 PM	83.42	1.668	0.857	1.95	2.18	50	230	0.765	0.084
SN22	Default	5/7/2015	12:01 PM	243.91	4.878	2.54	1.92	2.05	50	230	2.381	0.656
SN23	Default	5/7/2015	12:02 PM	365.7	7.314	3.644	2.01	2.31	50	230	3.169	0.07
SN24	Default	5/7/2015	12:03 PM	152.83	3.057	1.588	1.92	2.31	50	230	1.323	0.044
SN25	Default	5/7/2015	12:04 PM	160.41	3.208	1.617	1.98	2.35	50	230	1.363	0.038
SN26	Default	5/7/2015	12:06 PM	71.78	1.436	0.753	1.91	2.24	50	230	0.642	0.053
SN27	Default	5/7/2015	12:07 PM	244.35	4.887	2.454	1.99	2.35	50	230	2.076	0.027
SN28	Default	5/7/2015	12:08 PM	153.82	3.076	1.561	1.97	2.33	50	230	1.322	0.045
SN29	Default	5/7/2015	12:09 PM	192.01	3.84	1.975	1.94	2.24	50	230	1.711	0.108
SN30	Default	5/7/2015	12:10 PM	293	5.86	2.947	1.99	2.33	50	230	2.519	0.051
SN31	Default	5/7/2015	12:11 PM	321.32	6.426	3.337	1.93	2.32	50	230	2.765	0.085
SN32	Default	5/7/2015	12:12 PM	45.65	0.913	0.477	1.91	1.89	50	230	0.483	0.104

You can also see the raw nanodrop file here and the raw tab delimited table here.

**Update**
After reviewing the spec results from the Plate and the nanodrop results here. I have chosen the 40 samples to be sent to GBS. The samples from the Plate need to be renamed to have a P in front of the sample ID so that I can tell they were from the plate.

Samples from the Mollusk EZNA Kit in individual 1.5 ml tubes:

Sample ID	Number	Date	Time	ng/ul	A260	A280	260/280	260/230
HL10	1	5/7/2015	10:26 AM	96.08	1.922	0.966	1.99	2.2
HL11	2	5/7/2015	10:27 AM	192.3	3.846	1.951	1.97	2.28
HL12	3	5/7/2015	10:28 AM	58.12	1.162	0.584	1.99	2.15
HL14	4	5/7/2015	10:30 AM	127.92	2.558	1.275	2.01	2.35
HL16	5	5/7/2015	10:32 AM	98.46	1.969	0.978	2.01	2.3
HL17	6	5/7/2015	10:33 AM	73.78	1.476	0.724	2.04	2.32
HL18	7	5/7/2015	10:34 AM	66.94	1.339	0.668	2	2.22
HL19	8	5/7/2015	10:35 AM	287.52	5.75	2.813	2.04	2.34
HL2	9	5/7/2015	10:17 AM	141.47	3.537	1.749	2.02	2.27
HL20	10	5/7/2015	10:36 AM	144.94	2.899	1.412	2.05	2.33
HL21	11	5/7/2015	10:37 AM	356.18	7.124	3.63	1.96	2.35
HL22	12	5/7/2015	10:38 AM	222.98	4.46	2.234	2	2.39
HL23	13	5/7/2015	10:39 AM	393.05	7.861	4.097	1.92	2.33
HL24	14	5/7/2015	10:40 AM	60.32	1.206	0.62	1.95	2.11
HL25	15	5/7/2015	10:41 AM	152.2	3.044	1.549	1.96	2.3
HL25	16	5/7/2015	10:42 AM	142.1	2.842	1.41	2.02	2.67
HL25	17	5/7/2015	10:43 AM	128.42	2.568	1.29	1.99	2.29
HL26	18	5/7/2015	10:44 AM	336.54	6.731	3.345	2.01	2.41
HL27	19	5/7/2015	10:45 AM	377.31	7.546	3.813	1.98	2.32
HL28	20	5/7/2015	10:46 AM	103.32	2.066	1.07	1.93	2.24
HL29	21	5/7/2015	10:47 AM	74.49	1.49	0.768	1.94	2.2
HL3	22	5/7/2015	10:18 AM	58.05	1.451	0.716	2.03	2.19
HL30	23	5/7/2015	10:48 AM	274.08	5.482	2.804	1.95	2.26
HL31	24	5/7/2015	10:50 AM	94.38	1.888	0.947	1.99	2.18
HL32	25	5/7/2015	10:51 AM	269.6	5.392	2.786	1.94	2.16
HL4	26	5/7/2015	10:19 AM	119.39	2.985	1.504	1.98	2.27
HL5	27	5/7/2015	10:20 AM	64.32	1.608	0.805	2	2.2
HL6	28	5/7/2015	10:21 AM	78.93	1.973	0.976	2.02	2.22
HL7	29	5/7/2015	10:22 AM	115.35	2.884	1.419	2.03	2.3
HL9	30	5/7/2015	10:25 AM	172.45	3.449	1.726	2	2.31
NF1	1	5/7/2015	10:53 AM	330.81	6.616	3.396	1.95	2.32
NF10	2	5/7/2015	11:05 AM	124.73	2.495	1.255	1.99	2.22
NF11	3	5/7/2015	11:06 AM	447.26	8.945	4.552	1.97	2.25
NF12	4	5/7/2015	11:07 AM	93.36	1.867	0.963	1.94	2.08
NF13	5	5/7/2015	11:08 AM	277	5.54	2.847	1.95	2.18
NF14	6	5/7/2015	11:09 AM	298.5	5.97	2.983	2	2.32
NF15	7	5/7/2015	11:10 AM	155.67	3.113	1.569	1.98	2.3
NF16	8	5/7/2015	11:11 AM	135.04	2.701	1.396	1.94	2.15
NF18	9	5/7/2015	11:14 AM	292.28	5.846	2.973	1.97	2.21
NF19	10	5/7/2015	11:15 AM	90.2	1.804	0.934	1.93	2.25
NF2	11	5/7/2015	10:55 AM	210.64	4.213	2.199	1.92	2.35
NF20	12	5/7/2015	11:16 AM	374.23	7.485	3.772	1.98	2.36
NF21	13	5/7/2015	11:18 AM	233.53	4.671	2.416	1.93	2.37
NF22	14	5/7/2015	11:19 AM	86.59	1.732	0.893	1.94	2.35
NF23	15	5/7/2015	11:20 AM	204.91	4.098	2.067	1.98	2.35
NF24	16	5/7/2015	11:21 AM	231.38	4.628	2.391	1.94	2.34
NF25	17	5/7/2015	11:22 AM	126.3	2.526	1.384	1.82	1.59
NF26	18	5/7/2015	11:23 AM	159.12	3.182	1.597	1.99	2.31
NF27	19	5/7/2015	11:25 AM	101.21	2.024	1.058	1.91	2.33
NF28	20	5/7/2015	11:26 AM	128.03	2.561	1.279	2	2.33
NF29	21	5/7/2015	11:27 AM	176.44	3.529	1.786	1.98	2.33
NF3	22	5/7/2015	10:56 AM	242.37	4.847	2.514	1.93	2.21
NF30	23	5/7/2015	11:28 AM	187.06	3.741	1.895	1.97	2.31
NF31	24	5/7/2015	11:29 AM	117.53	2.351	1.179	1.99	2.27
NF32	25	5/7/2015	11:30 AM	272.95	5.459	2.808	1.94	2.28
NF4	26	5/7/2015	10:57 AM	207.32	4.146	2.201	1.88	2
NF5	27	5/7/2015	10:58 AM	323.45	6.469	3.368	1.92	2.11
NF6	28	5/7/2015	10:59 AM	317.97	6.359	3.327	1.91	2.35
NF6	29	5/7/2015	11:00 AM	270.17	5.403	2.794	1.93	2.38
NF7	30	5/7/2015	11:01 AM	191.55	3.831	1.985	1.93	2.27
NF8	31	5/7/2015	11:02 AM	361.84	7.237	3.677	1.97	2.33
NF9	32	5/7/2015	11:03 AM	166.86	3.337	1.677	1.99	2.31
SN1	1	5/7/2015	11:36 AM	107.82	2.156	1.096	1.97	2.16
SN10	2	5/7/2015	11:46 AM	118.82	2.376	1.221	1.95	2.21
SN11	3	5/7/2015	11:47 AM	267.08	5.342	2.724	1.96	2.32
SN12	4	5/7/2015	11:49 AM	127.94	2.559	1.317	1.94	2.32
SN13	5	5/7/2015	11:50 AM	317.61	6.352	3.194	1.99	2.32
SN14	6	5/7/2015	11:51 AM	146.97	2.939	1.498	1.96	2.23
SN15	7	5/7/2015	11:52 AM	164.33	3.287	1.665	1.97	2.24
SN17	8	5/7/2015	11:54 AM	125.5	2.51	1.293	1.94	2.24
SN18	9	5/7/2015	11:56 AM	170.94	3.419	1.748	1.96	2.35
SN2	10	5/7/2015	11:37 AM	278.11	5.562	2.803	1.98	2.26
SN20	11	5/7/2015	11:59 AM	104.44	2.089	1.078	1.94	2.33
SN21	12	5/7/2015	12:00 PM	83.42	1.668	0.857	1.95	2.18
SN22	13	5/7/2015	12:01 PM	243.91	4.878	2.54	1.92	2.05
SN23	14	5/7/2015	12:02 PM	365.7	7.314	3.644	2.01	2.31
SN24	15	5/7/2015	12:03 PM	152.83	3.057	1.588	1.92	2.31
SN25	16	5/7/2015	12:04 PM	160.41	3.208	1.617	1.98	2.35
SN27	17	5/7/2015	12:07 PM	244.35	4.887	2.454	1.99	2.35
SN28	18	5/7/2015	12:08 PM	153.82	3.076	1.561	1.97	2.33
SN29	19	5/7/2015	12:09 PM	192.01	3.84	1.975	1.94	2.24
SN3	20	5/7/2015	11:38 AM	122.94	2.459	1.255	1.96	2.27
SN30	21	5/7/2015	12:10 PM	293	5.86	2.947	1.99	2.33
SN31	22	5/7/2015	12:11 PM	321.32	6.426	3.337	1.93	2.32
SN4	23	5/7/2015	11:39 AM	225.29	4.506	2.236	2.01	2.31
SN5	24	5/7/2015	11:41 AM	89.6	1.792	0.914	1.96	2.21
SN7	25	5/7/2015	11:43 AM	107.8	2.156	1.117	1.93	2.2
SN8	26	5/7/2015	11:44 AM	212.99	4.26	2.185	1.95	2.25
SN9	27	5/7/2015	11:45 AM	115.94	2.319	1.206	1.92	2.29
SN6	28	5/7/2015	11:42 AM	79.5	1.59	0.808	1.97	2.23
SN16	29	5/7/2015	11:53 AM	77.75	1.555	0.821	1.89	1.87

Samples from the DNEasy Plate:

Sample	ng/ul	plate designation	number
PHL04	76.40	D1	1
PHL15	71.58	G2	2
PHL18	86.78	B3	3
PHL21	80.37	E3	4
PHL24	73.51	H3	5
PHL26	72.73	B4	6
PHL27	103.28	C4	7
PHL31	79.00	G4	8
PHL14	69.76	F2	9
PHL20	59.87	D3	10
PNF02	77.88	B5	1
PNF07	83.26	G5	2
PNF19	91.79	C7	3
PNF22	73.80	F7	4
PNF29	76.51	E8	5
PNF32	79.92	H8	6
PNF04	68.87	D5	7
PNF25	67.80	A8	8
PSN03	83.00	C9	1
PSN07	97.94	G9	2
PSN09	79.18	A10	3
PSN12	80.85	D10	4
PSN13	78.33	E10	5
PSN22	77.95	F11	6
PSN23	92.72	G11	7
PSN27	83.17	C12	8
PSN28	90.17	C13	9
PSN29	82.61	C14	10
PSN30	85.23	C15	11

5 6 2015 qPCR Primer Dimer check

Yesterday I ran another qPCR on both sets of isolated RNA to determine whether the samples were truly contaminated with gDNA or if they were producing primer dimers. This required that we look at the melt curve to determine the size of the products being created.

The qPCR reagent table:

	Volume	Reactions X12
Ssofast Evagreen MM	10	280
FWD Primer	0.5	14
REV Primer	0.5	14
Nuclease Free H2O	8.5	238
RNA	0.5

For the qPCR I used Actin primers and a positive control from Fidalgo seed oysters extracted on 3/23/2015 with a concentration of 167.3 ng/ul. My no template control contained no DNA as to show there was no contamination in the Master Mix.

qPCR protocol:

1. Added each from greatest volume to least to make the master mix.

2. Pipetted 19.5 ul master mix into each well of a qPCR partial plate

3. Added 0.5 ul sample to each tube

Table Layout.

9	10	11	12
C-	C+	C-	C+
42715HM1	42715SM1	42715HT1	42715ST1
42715HM2	42715SM2	42715HT2	42715ST2
42715HM3	42715SM3	42715HT3	42715ST3
42715HM4	42715SM4	42715HT4	42715ST4
42715HM5	42715SM5	42715HT5	42715ST5

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Read
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

You can see the amplification curves below:

You can see the Melt curve below:

It appears that there is definite gDNA contamination in the samples which is a shame. To remedy this I will be running another round of DNase treatment on them today. I'll check them with another qPCR tonight. You can view the raw qPCR file here.

Tuesday, May 5, 2015

5 5 2015 310 Heat Shock RNA Isolation / qPCR

Today I process the heat shock samples from the Fish 310 lab. I isolated the RNA, DNase treated the sample, and ran a qPCR for quality control. I changed a few things from the previous protocols to make things run smoother and more efficient today. I've also taken several of Sam's suggestions on how I can improve my notebook.

RNA Isolation Protocol.

100 mg tissue homogenized in 1 ml RNAzol RT at room temp.
Added 400 ul 0.1% DEPC water to homogenate.
Incubated 15 minutes at room temp
Centrifuged 15 minutes at 11,400 rpm (~12,000 g)
Transferred 1 ml supernatant to fresh tube
Added 400 ul 75% EtOH
Mixed via inversion for 15 seconds
Incubated samples 10 minutes at room temp
Centrifuged 8 minutes at 11,400 rpm (~12,000 g)
Discarded supernant
Added 600 ul 75% EtOH
Vortexed 15 seconds
Centrifuged 3 minutes at 7,400 rpm (~8000 g)
Repeated steps 10 - 13.
Removed supernatant
resuspended RNA in 50 ul 0.1% DEPC water

Instead of nanodropping the RNA, I immediately Turbo DNase Treated the samples to remove any DNA contamination.

DNase Treatment Protocol:

1. Added 5 ul DNase 10X Buffer to each 50 ul of RNA.
2. Added 1 ul Turbo DNase to each RNA.
3. Incubated at room temp for 30 minutes
4. Added 5 ul DNase inhibitor to each sample
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 9400 rpm (~10,000 g) for 1.5 minutes
7. Decanted the top 50 ul of supernatant to fresh tube

8. Nanodropped

Nanodrop Results (including positive control):

Sample ID	Date	Time	ng/ul	A260	A280	260/280	260/230
C+	5/5/2015	4:17 PM	167.3	4.182	2.092	2	2.34
42715HT1	5/5/2015	4:18 PM	238.58	5.965	3.366	1.77	0.66
42715HT2	5/5/2015	4:19 PM	422.45	10.561	5.431	1.94	1
42715HT3	5/5/2015	4:21 PM	175.57	4.389	2.26	1.94	1.79
42715HT4	5/5/2015	4:22 PM	251.19	6.28	3.532	1.78	0.68
42715HT5	5/5/2015	4:23 PM	391.29	9.782	5.133	1.91	0.87
42715ST1	5/5/2015	4:24 PM	690.94	17.274	10.258	1.68	0.49
42715ST2	5/5/2015	4:25 PM	393.97	9.849	5.181	1.9	0.91
42715ST3	5/5/2015	4:25 PM	365.73	9.143	4.917	1.86	0.99
42715ST4	5/5/2015	4:26 PM	426.33	10.658	5.407	1.97	0.99
42715ST5	5/5/2015	4:27 PM	343.49	8.587	4.622	1.86	0.81

For the qPCR I used Actin primers and a positive control from Fidalgo seed oysters extracted on 3/23/2015. My negative control contained no DNA as to show there was no contamination in the Master Mix. I also bumped up the volume of sample used to 0.5 ul due to issues with the pipetter not being able to accurately pipette 0.2 ul. This is about 2.5 X the concentration of contamination gDNA that the cDNA will have in it. I also bumped down the amount of water by 1 ul per reaction so as to ensure proper reaction mixture ratios were maintained.

qPCR Reagent Table:

	Volume	Reactions X12
Ssofast Evagreen MM	10	140
FWD Primer	0.5	7
REV Primer	0.5	7
Nuclease Free H2O	8.8	123.2
RNA	0.5

qPCR protocol:

1. Added each from greatest volume to least to make the master mix.

2. Pipetted 20 ul master mix into each well of a qPCR partial plate

3. Added 0.5 ul sample to each tube

qPCR plate lay out:

	11	12
C	C-	C+
D	42715HT1	42715ST1
E	42715HT2	42715ST2
F	42715HT3	42715ST3
G	42715HT4	42715ST4
H	42715HT5	42715ST5

This layout can be seen in the top left corner of the qPCR image below.

qPCR program:

Sybr New Plate+Sybr cDNA 55 melt 1 Read
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

Results:

It looks like the DNase treatment removed some but not all the DNA. The DNA replicates much much earlier in the cycles. Overall I think these samples are good samples with high concentrations and mild DNA contamination. You can see the data files for the qPCR here and here.

Sometime this week I will begin the process of creating cDNA to use with qPCR to test for HSP70 and other RNA markers.

Monday, May 4, 2015

5 4 2015 DNase treatment

Today I ran the DNase treatment on the samples to remove any excess DNA from the samples. The treatment was pretty simple but I don't think it helped much as their was amplification at roughly the sample cycle as there was in the last quality check. The DNase treatments were old but I don't think that would cause an issue. I also had to mix kits as there wasn't enough reagents in a single kit to do the treatments on 10 samples.

Protocol for treatment:

1. Added 10 ul DNase 10X Buffer to each 100 ul of RNA.
2. Added 2 ul Turbo DNase to each RNA.
3. Incubated at 37 C for 30 minutes
4. Added 10 ul DNase inhibitor to each sample
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 10,000 g for 1.5 minutes
7. Decanted the top 100 ul of supernatant to fresh tube
8. Placed in -80 until I could run qPCR

Later this afternoon I ran the qPCR on the treatment.

Reagent work sheet:

	Volume	Reactions X12
Ssofast Evagreen MM	10	140
FWD Actin Primer	0.5	7
REV Actin Primer	0.5	7
Nuclease Free H2O	9.8	137.2
RNA	0.2

1. Added each from greatest volume to least to make the master mix.

2. Pipetted 21 ul master mix into each well of a qPCR partial plate

3. Added 0.2 ul sample to each tube

4. Positive control was DNA extract from Oly seed oysters

5. Negative control had nothing added to the master mix

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Read
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

You can see the results below

bright green = positive control, red = negative control, light blue down to brown = HM1-5, dark blue to yellow = SM1-5

The positive control began amplifying at the 21 cycle marks. One sample began at 30 cycles the rest were between 34 and 36.

You can see the raw datafile here.

Tomorrow I will isolate the RNA from the 310 heat shock samples, treat with DNase, and qPCR for quality.

Friday, May 1, 2015

May 2015 Goals

In an effort to keep myself on track to compete the SAFS masters program by later this year I am going to write monthly goals to complete for each month.

This month:

Process samples to produce cDNA for qPCR
Run qPCR using primers for HSP70, Actin, and other interesting primers
Edit the Materials and Methods section
Write the Results sections of my next manuscript
Begin writing the Intro/Discussion
Prepare for my wedding

5 1 2015 qPCR Quality Check

Today I ran a quality check on the samples that I isolated yesterday. Instead of creating cDNA before doing the qPCR I ran just the RNA with Actin primers to determine if there was any genomic contamination in the RNA samples. I diluted the RNA to levels similar to that which would have gone from the cDNA process to the qPCR process. This was to ensure that any genomic dna would be at levels similar to that from from the cDNA process and wouldn't over estimate the concentration of them in the sample.

The qPCR protocol was as follows

Master Mix

	Volume	Reactions X12
Ssofast Evagreen MM	10	120
FWD Primer	0.5	6
REV Primer	0.5	6
Nuclease Free H2O	9.8	117.6

1. Added the reagents from highest volume to lowest to the master mix

2. Vortexed

3. pipetted into .5 ml qPCR tubes

4. Added 0.2 ul RNA to each tube from each isolation

5. Placed into the Opticon 2 qPCR machine

6. Ran the program "Sybr New Plate + Sybr cDNA 55 melt 2 reads"

Results

As you can see, the samples began amplifying at 27-30 cycles which is indicative of a genomic DNA contamination. The negative control did not begin amplifying until 37 cycles.

For future work, I need to use a DNase treatment to remove genomic DNA before I create cDNA. Next week I will isolate the 310 heat shock animals, DNase treat everything, and produce cDNA.