Protocol for PocHistone Amplification and Test Sanger Sequencing Prep

Goal:

Amplify the histone 3 region of Pocillopora coral DNA using PCR (polymerase chain reaction). Then use amplification product for Sanger Sequencing and determination of Pocillopora coral species between P. eydouxi and P. meandrina on 4 test samples out of 32 samples. Primer sequences and concept developed in Johnston et. al 2018.

Information on Sanger sequencing at the URI GSC is here. Sequencing takes place after 10am on Tuesdays and Thursdays.

Process

Followed the PocHistone Protocol exactly. See that for in depth details for this protocol.

DNA Dilutions 2020-08-27

DNA dilutions for samples completed by Maggie on 2020-08-27. See the notebook post for in depth details for this protocol.

Histone Amplifications 2021-01-25

• 32 samples plus three negative controls is 35 reactions, use an additional 3 for error
• Made a master mix for 38 samples and 3 reactions each:
  • 1900ul Phusion master mix
  • 49.4ul working stock PocHistoneF 10uM Primer
  • 49.4ul working stock PocHistoneR 10uM Primer
  • 1672ul nuclease free water
• Added 97ul of master mix into 35 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 PocHistone program

1X Bead Cleanup and Quantification 2021-01-25

• 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)

Broad Range Qubit 2020-01-25

• dsDNA broad range Qubit assay for 35 samples (n# 40)
• Followed qubit protocol
• Included 32 samples, 3 controls, and 2 standards

Sample.ID	Qubit reading 1 (ng/ul)	Qubit reading 2 (ng/ul)	average DNA (ng/ul)
std. 1	176.98	NA	NA
std. 1	20368.25	NA	NA
E10	49	49	49
C22	38.2	38.4	38.3
E10	49	49	49
E2	49	49.4	49.2
C19	44.6	44.6	44.6
C25	49.6	49.6	49.6
C29	47.8	47.8	47.8
C21	51.8	50.8	51.3
E4	48.6	49.2	48.9
E12	52.8	53.8	53.3
E1	48	47.8	47.9
C27	52.6	53.8	53.2
E6	49.2	49.2	49.2
E8	44.8	45.4	45.1
C20	54	54	54
C28	52.6	52.2	52.4
E9	54.4	54.4	54.4
E14	52.2	51.8	52
E15	49.6	48.4	49
C17	52.6	53	52.8
E3	47.6	47.6	47.6
C30	55.2	55.2	55.2
C24	45.8	45.6	45.7
E16	48	47.6	47.8
C31	38.4	38	38.2
E7	44.8	45.4	45.1
C23	43.6	43.6	43.6
E11	45.8	45.8	45.8
E13	40	40	40
C18	35	35.2	35.1
E5	43.6	43.8	43.7
C32	33	33.4	33.2
C26	32.8	32.8	32.8

1% Gel To Confirm Bands 2021-01-26

• Made a 1% gel in the medium gel box
• Bands are strong but some smearing is noticeable which could be an issue. It’s not contamination because our controls are blank. So it’s either an imaging artifact or something that amplified non-specific in the sample. We will be sequencing four samples from a sample size of each treatment and one with smearing and one without to see if there is more of a problem that needs troubleshooting.

Dilution and Sequencing Prep 2021-01-27

• Used samples E12 (small band underneath); E8 (no smearing); C17 (small band above); C30 (smearing)
• For sequencing I need 9ul (4 * 2 +1 for error) of 3.2uM PocHistone R primer
• The PocHistone product should be ~669 bp, so 669 / 100 * 1.25 * 2 = 16.7ng of each sample
• 16.7ng is still less than 1ul for every sample, so I did a 1:10 dilution for each one
• Added 18ul of ultra pure water to 4 new strip tubes, and added 2ul of each cleaned DNA to their corresponding well
• Pipetted 16.7ng of each sample from diluted stock to new strip tubes
• Increased volume in tubes to 10ul with ultra pure water

Sample	GSC sample code	Dilution conc.	ng needed	Volume diluted PCR product	volume ultra pure H2O
E12	HPD33	5.33	16.7	3.13	6.87
E8	HPD34	4.51	16.7	3.70	6.30
C17	HPD35	5.28	16.7	3.16	6.84
C30	HPD36	5.52	16.7	3.03	6.97

• Made 9ul of diluted 10uM stock PocHistone R to 3.2uM
• Added 6.12ul of ultra pure water to 1 PCR tube
• Added 2.88ul of 10uM PocHistone R primer to tube
• Vortex and spin down to mix
• Added 2ul of 3.2uM primer to each of the 8 strip tubes diluted for sequencing
• Stored prepared samples in -20 freezer

Sequencing Submission 2021-01-28

• Submitted four test samples for sequencing at 9:40am

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
HPD33		PCR	669	5.33	16.725	3.13	6.87	2
HPD34		PCR	669	4.51	16.725	3.70	6.30	2
HPD35		PCR	669	5.28	16.725	3.16	6.84	2
HPD36		PCR	669	5.52	16.725	3.03	6.97	2

Sequences Recieved 2021-01-29

• All sequences looked good, was able to align and delinate to species using Geneious Prime.