mtORF Amplification and Sanger Sequencing Prep for the Pocillopora Samples from the Mo’orea Connect Project

Goal: Amplify the 56 samples, with 4 control reactions for mtORF. Then dilute them properly and send them to the GSC for Sanger sequencing.
Major Results: Great amplifications again with clear bands and no contamination.
Major Takeaways: The protocol works great for even more samples (60). Qubiting that many was a hassle though, and maybe a plate reader quantification system would be better. It might be good to increase the resuspension volume after the bead clean, however I still fell like with the variability of the qubit values after the clean up that even so some samples would need to be diluted. And it would be more of a hassle to only have to dilute random samples than to have to dilute all samples.

Process

Followed the mtORF amplification protocol exactly. See that for in depth details for this protocol

DNA Dilutions 2020-08-25

• Arranged samples in a 96 well plate (unfortunately in numerical order, because I was doing them all at the same time it seemed fine)
• Allocated DNA for 10ng/ul in 20ul of nuclease free water, so 200ng of DNA. If samples had concentrations below 10ng/ul to start with, just 10ul of those samples was added to each well

mtORF Amplifications 2020-09-03

• 56 samples plus four negative controls is 60 reactions, use an additional 5 for error
• Made a master mix for 65 samples and 3 reactions each:
  • 3250ul Phusion master mix
  • 84.5ul FatP6.1 10uM Primer
  • 84.5ul RORF 10uM Primer
  • 2860ul nuclease free water
• Added 97ul of master mix into 60 wells in a new plate
• Used a multichannel to add 3ul of DNA from the dilution plate in to the same orientation wells in the plate with the master mix
• Covered plate and vortexed and spun down
• Separated plate out 2 times into 3 separated reaction mixes each with 33ul
• Covered plates, spun down, and placed in three thermocyclers FatP6.1 RORF program

Layout

1X Bead Cleanup 2020-09-04

• Combined triplicate reactions back together
• Added 1X (100ul) beads to each well
• Followed bead cleanup protocol
• Resuspended and eluted DNA in 50ul ultra-pure water and removed into a new plate (same orientation)

Gel and Quantification 2020-09-08

• Made a 1% gel in the medium gel box
• One band at ~1000bp, exactly right! No contamination, only imaging artifacts
• dsDNA broad range Qubit assay for 60 samples (n# 67)

Sample.ID	Qubit reading 1 (ng/ul)	Qubit reading 2 (ng/ul)	average DNA (ng/ul)
35	32.2	32.6	32.4
38	28	27.6	27.8
41	28	28.2	28.1
42	28.4	28.4	28.4
45	22.2	22.2	22.2
46	27.2	27	27.1
47	32	32.2	32.1
48	28.8	29	28.9
49	30.8	30.8	30.8
50	28.6	28	28.3
59	30.2	30.2	30.2
60	39.4	40	39.7
61	47	47.6	47.3
62	40.4	40.8	40.6
65	38.4	38	38.2
157	37.2	37.2	37.2
158	40.2	40.4	40.3
159	37.2	37.8	37.5
160	40	40.2	40.1
161	43.8	43.6	43.7
164	39	39.4	39.2
165	37	37.6	37.3
167	42.6	42.6	42.6
168	36.6	36.4	36.5
169	36.2	35.6	35.9
171	33.2	33.6	33.4
172	29.2	29.4	29.3
174	27.8	28.2	28
175	26	26.4	26.2
176	28	28	28
177	27.6	27.2	27.4
178	27.2	27.6	27.4
182	30.4	30.6	30.5
183	29.8	29.8	29.8
184	48.4	48.4	48.4
185	37	37.4	37.2
186	42.4	42.8	42.6
188	42.2	42	42.1
189	37	37	37
190	38.6	39	38.8
191	40	40.4	40.2
192	37	37.4	37.2
217	39.4	39.6	39.5
224	36.2	36.4	36.3
225	32.4	33.2	32.8
232	30.6	31	30.8
234	35.2	35.2	35.2
236	34	34	34
237	51.4	51.4	51.4
238	52.8	53.2	53
239	45	45.8	45.4
240	49.2	49.4	49.3
241	46	46.6	46.3
243	41.8	42.4	42.1
244	41.2	41.2	41.2
245	37.6	37.6	37.6

Dilution and Sequencing Prep 2020-09-09

• Diluted each DNA amplification by 1:10 (2ul of DNA and 20ul of ultra-pure water)
• Created plate with 25ng of DNA for each sample and ultra-pure water up to 10ul
• Added 2ul of 3.2uM RORF primer to each well
• Covered, vortexed, spun down plate
• Brought upstairs for sequencing the next day

Spreadsheet for Sequencing

Sample IDa	Well (GSC use only)	Template Typeb	A. Template Size (bases)	B. Template Stock Conc. (ng/µl)	C. PCR template: ng needed = ((A ÷ 100) x 1.25) x 2	D. PCR template: Volume = (C ÷ B) µl	F. Volume PCR-H20 needed (10 minus D or E) µl	G. Volume primer needed 1 µl per reaction
HPT1		PCR	1000	3.24	25	7.7	2.3	2
HPT2		PCR	1000	2.78	25	9.0	1.0	2
HPT3		PCR	1000	2.81	25	8.9	1.1	2
HPT4		PCR	1000	2.84	25	8.8	1.2	2
HPT5		PCR	1000	2.22	25	10.0	0.0	2
HPT6		PCR	1000	2.71	25	9.2	0.8	2
HPT7		PCR	1000	3.21	25	7.8	2.2	2
HPT8		PCR	1000	2.89	25	8.7	1.3	2
HPT9		PCR	1000	3.08	25	8.1	1.9	2
HPT10		PCR	1000	2.83	25	8.8	1.2	2
HPT11		PCR	1000	3.02	25	8.3	1.7	2
HPT12		PCR	1000	3.97	25	6.3	3.7	2
HPT13		PCR	1000	4.73	25	5.3	4.7	2
HPT14		PCR	1000	4.06	25	6.2	3.8	2
HPT15		PCR	1000	3.82	25	6.5	3.5	2
HPT16		PCR	1000	3.72	25	6.7	3.3	2
HPT17		PCR	1000	4.03	25	6.2	3.8	2
HPT18		PCR	1000	3.75	25	6.7	3.3	2
HPT19		PCR	1000	4.01	25	6.2	3.8	2
HPT20		PCR	1000	4.37	25	5.7	4.3	2
HPT21		PCR	1000	3.92	25	6.4	3.6	2
HPT22		PCR	1000	3.73	25	6.7	3.3	2
HPT23		PCR	1000	4.26	25	5.9	4.1	2
HPT24		PCR	1000	3.65	25	6.8	3.2	2
HPT25		PCR	1000	3.59	25	7.0	3.0	2
HPT26		PCR	1000	3.34	25	7.5	2.5	2
HPT27		PCR	1000	2.93	25	8.5	1.5	2
HPT28		PCR	1000	2.8	25	8.9	1.1	2
HPT29		PCR	1000	2.62	25	9.5	0.5	2
HPT30		PCR	1000	2.8	25	8.9	1.1	2
HPT31		PCR	1000	2.74	25	9.1	0.9	2
HPT32		PCR	1000	2.74	25	9.1	0.9	2
HPT33		PCR	1000	3.05	25	8.2	1.8	2
HPT34		PCR	1000	2.98	25	8.4	1.6	2
HPT35		PCR	1000	4.84	25	5.2	4.8	2
HPT36		PCR	1000	3.72	25	6.7	3.3	2
HPT37		PCR	1000	4.26	25	5.9	4.1	2
HPT38		PCR	1000	4.21	25	5.9	4.1	2
HPT39		PCR	1000	3.7	25	6.8	3.2	2
HPT40		PCR	1000	3.88	25	6.4	3.6	2
HPT41		PCR	1000	4.02	25	6.2	3.8	2
HPT42		PCR	1000	3.72	25	6.7	3.3	2
HPT43		PCR	1000	3.95	25	6.3	3.7	2
HPT44		PCR	1000	3.63	25	6.9	3.1	2
HPT45		PCR	1000	3.28	25	7.6	2.4	2
HPT46		PCR	1000	3.08	25	8.1	1.9	2
HPT47		PCR	1000	3.52	25	7.1	2.9	2
HPT48		PCR	1000	3.4	25	7.4	2.6	2
HPT49		PCR	1000	5.14	25	4.9	5.1	2
HPT50		PCR	1000	5.3	25	4.7	5.3	2
HPT51		PCR	1000	4.54	25	5.5	4.5	2
HPT52		PCR	1000	4.93	25	5.1	4.9	2
HPT53		PCR	1000	4.63	25	5.4	4.6	2
HPT54		PCR	1000	4.21	25	5.9	4.1	2
HPT55		PCR	1000	4.12	25	6.1	3.9	2
HPT56		PCR	1000	3.76	25	6.6	3.4	2