2025-09-05 Sanger Sequencing Preps for Moorea E5 Project Acropora sp.

The samples processed today are of Acropora species. Total of 50 colonies of Acropora were collected as a part of E5 Moorea Project. Here, I am processing the gDNA samples extracted from these 50 colonies for Sanger sequencing to identify species within these genera.

This post is also adapted based on the procedure from Arianna Huffmyer Notebook Post.

Overview

gDNA extractions for 44 of the colonies were completed by Kristina Terpis in 2021 and 2022. Some of the gDNA extractions from six colonies were sent off to Azenta for sequencing. Hence, Hollie Putnam extracted gDNA for those colonies on 08/29/2025.

My samples were included in different boxes which were extracted by Kristina. The gDNA samples I used for the colonies are below. The Master Molecular Sample List is here while the Acropora colony IDs for the master timeseries data is here.

Protocols Used

Acropora

Sample Information

Add sheet of all the samples you selected and their timepoints.

Primer selection

Hollie and Arianna have previously talked about the primers to use for Acropora species identification. Hollie previously tested some of the primers from the literature. Arianna have used both the PaxC (PaxC_intron_FP1 forward and PaxC_intron_RP1 reverse) and CRf/CO3r (CRF forward and CO3r reverse) which were avaliable in the lab previously while prepping Acropora samples in 2024.

This time, Hollie recommended to use the CRf/CO3r (CRf forward and CO3r reverse) primer. |

Crf/CO3r sequencing

2025-09-05

  • Mitochondrial putative control (933+ bp) + 83 bp of cytochrome oxidase III as in Vollmer et al. 2002

The primers were hydrated but were present in their original concentration which was 100μM each. Hence, I diluted the primers as required for the Master mix to 10μM in two tubes.

Component Volume of Primer (μL) Volume of Nuclease Free Water (μL) Final Concentration of the product (μM)
CRf Primer 100μM 10μL 90μL 10μM
CO3r Primer 100μM 10μL 90μL 10μM

The diluted primers are stored in the tubes in the same “Putnam Lab Stick Primer Transparent Box on Shelf -20 C Freezer F” along with the concentrated primers.

I prepared the master mix using OneTaq Quick-Load 2X Master Mix from New England Biolabs. I made the master mix for 60 rxns as I was doing PCR for the first time.

Component rxns 60 rxns
OneTaq Quick-Load 2X master mix 12.5 μL 750 μL
10 μM CRf Primer 0.5 μL 30 μL
10 μM CO3r Primer 0.5 μL 30 μL
Nuclease Free Water 10.5 μL 630 μL
DNA Template 1μL  
  25 μL rxns 24 μL rxns

I prepared one single PCR plate containing 52 samples; 50 of which were gDNA extracted from 50 Acropora colonies by Kristina in 2021-2022 and Hollie in 2025.

I used a Danielle Becker Heatwave Acropora pulchra as a postive control (no. 24 gDNA from 10/29/23). Water was used as a negative control in the plate.

The PCR plate maps for this PCR shows the tube number followed by colony ID of the sample collected.

The PCR Map for this day is below

PCR MAP.

As the gDNA samples were present in the tubes and not plated on gDNA plates like before, I directly plated the samples onto the 96 well plates for PCR.

The PCR protocol (“Acro” on Putnam Account) was used similar to previous Acropora PCRs by Arianna.

The settings for the thermocycler is follows:

  • 95°C for 3 min [ cycle]
  • 94°C for 30 sec, 53°C for 30 sec, 72°C for min [35 cycles] Note: Melting temperature chosen based on lowest primer melting temperature
  • 72°C for 5 min
  • Hold at 4°C

After PCR, samples were stored in the 4 C fridge.

I than ran two gels i.e. One Small and One Medium for the CRf/CO3r primer set using 2% gel at 80V for 90 min. I used only one DNA ladder of KB for the same.

For 2% Gel

Gel Format 1X TAE BUffer (mL) Agarose (in g)
Small Box 75 mL .5 g
Medium Box 100 mL 2.0 g
Large Box 150 mL 3.0 g

Sample loaded in each gel is 4μL.

Only one DNA ladder 1KB was used for these gels as previously 100 bp ladder was also used by Arianna. The 100 bp ladder was not avaliable and as the targeted band size expected was closer to a 1000 bp, I went forward to just use the 1KB DNA Ladder.

ACR CRf/CO3r Gel ACR CRf/CO3r Gel 2

Results from the Gel

  1. Out of the 50 Colonies for Acropora, two colonies which are ACR-140 and ACR-180 did not show any bands signifying that the PCR amplification did not work for those two.
  2. The gel products look nice however have a weird shape for all the bands. After consulting with Hollie, I realized that there could be too much product hence later for the next PCRs I loaded on 3μL of sample for running the gel on her recommendation.
  3. There were no bands in the negative controls and band in the positive control for A. pulchra. All other samples look good.
  4. As the two samples from the colonies failed I would be redoing the PCR again with dilutions as well as the original concentration using the same tube numbers from timepoint .

2025-09-10

For today, I redid PCR for two colonies that did not show any band for the gel ran on 2025-09-05. The two colony_ids with no band were ACR-140 and ACR-180.

I also made a new master mix today.

Component rxns 16 rxns
OneTaq Quick-Load 2X master mix 12.5 μL 200 μL
10 μM CRf Primer 0.5 μL 8.0 μL
10 μM CO3r Primer 0.5 μL 8.0 μL
Nuclease Free Water 10.5 μL 168 μL
DNA Template 1μL  
  25 μL rxns 24 μL rxns

I am re-running the PCR using an 8-strip tube. For each colony, I’ve prepared three reactions corresponding to:

  1. The original sample (same concentration as before)

  2. 1:10 dilution

    Sample ID Volume of Sample Volume of Water
    T-207 ACR-140 10 μL 90 μL
    T-197 ACR-180 10 μL 90 μL

  3. 1 :100 dilution

    Sample ID Volume of Sample Volume of Water
    T-207 ACR-140 .0 μL 99.0 μL
    T-197 ACR-180 .0 μL 99.0 μL

In total, I’m including three samples per colony. I used a Danielle Becker Heatwave Acropora pulchra as a postive control (no. 24 gDNA from 10/29/23). Water was used as a negative control in the plate.

The PCR Map for this day is below

PCR MAP.

Samples were stored in the fridge for the next steps.

The same setting was run for the PCR again and I ran only one 2% gel at 80 V for 90 min for 8 samples.

This time I only loaded 3μL of sample and used the same DNA ladder 1KB.

ACR CRf/CO3r Gel 09-10-2025

Results from the Gel

  1. Out of the two colonies for Acropora, ACR-140 did not show any bands even with dilutions.
  2. The gel products look nice for colony_id ACR-180 and the bands looks more linear now.
  3. There were no bands in the negative controls and band in the positive control for A. pulchra.
  4. As the one samples from the ACR-140 colony failed I would be redoing the PCR again with other samples from different timepoints.

2025-09-11

Today’s goal was to redo the ACR-140 colony_id’s remaining timepoints to check if the DNA is amplified for further process. There were total three more timepoints (TP2, TP3 and TP4) however I could only find tubes for TP2 and TP3 hence moved forward with only processing those two.

Sample Information

Tube Number Colony_ID Timepoint
467 ACR-140 TP2
675 ACR-140 TP3

The Master mix for today goes as follows: |Component| rxns|8 rxns| |———|———|———-| |OneTaq Quick-Load 2X master mix | 12.5 μL | 100 μL | |10 μM CRf Primer| 0.5 μL|4.0 μL| |10 μM CO3r Primer| 0.5 μL|4.0 μL| |Nuclease Free Water|10.5 μL|84 μL| |DNA Template| 1μL | | || 25 μL rxns |24 μL rxns|

In total, I’m including two samples for each timepoint ACR-140 colony. I used a Danielle Becker Heatwave Acropora pulchra as a postive control (no. 24 gDNA from 10/29/23). Water was used as a negative control in the plate.

The PCR Map for this day is below

PCR MAP.

The same setting was run for the PCR again and I ran only one 2% gel at 80 V for 90 min for 4 samples.

This time sample loaded was 3.0 μL and used the same DNA ladder 1KB.

Samples were stored in the fridge for the next steps after loading.

ACR CRf/CO3r Gel 09-11-2025

Results from the Gel

  1. The gel products look nice for colony_id ACR-140 and the bands looks more linear now.
  2. There were no bands in the negative controls and band in the positive control for A. pulchra.

As by the end of DAY 3, the PCR worked for all the 50 colonies of Acropora.

For all the 50 colonies from a single timepoint of the Acropora sp worked, we can now proceed with cleaning and preparation for sequencing for all the single timepoint for all the 50 Acropora colonies.

I then perfomed a clean up using the Zymo DNA Clean & Concentrator-5 50 Preps Kit in single tube format.

NOTE: During this cleaning process, I removed the failed samples from the first round of PCR and replaced with the successful PCR product for those samples. For ACR-140, I used the Tube# 467 from Timepoint 2 and that for ACR-180, I chose Tube # 197 with successful PCR product.

As the kit was not opened previously, I added 24 mL 100% Ethanol to the 6mL DNA Wash Buffer concentrate. I also diluted the forward and reverse primer to 3.2µM.

For 50 colonies, I made enough diluted primer that I can use it for the sequencing plate. Final Volume= 200 μL

Component Volume of Water μL Volume of Primer μL Final Concentration μM
100 μM CRf Primer 193.6 μL 6.4 μL 3.2 μM
100 μM CO3r Primer 193.6 μL 6.4 μL 3.2 μM

The steps for the cleanup goes as follows:

  1. Spin the PCR Product plate in the centrifuge or the rotator.

  2. Take .5 mL of microcentrifuge tubes and add DNA Binding Buffer in 5: ratio to the PCR product.

    Note: As some of my samples were not exactly 20 μL in volume I had to do adjustments. I added the DNA buffer in 5: ratio as per below calculations.

    Volume of DNA Binding Buffer in μL PCR Product Volume in μL
    35 uL 7 μL
    75 μL 15 μL
    85 μL 17 μL
    100 μL 20 μL
  3. Vortex the mixture or mix it with the pipette so that it is completely mixed.
  4. Transfer the mixture to a provided Zymo-Spin COlumn in a collection tube.
  5. Centrifuge for min at 13,000 rcf. Discard the flow-through in a beaker for now.

  6. Add 200 μL of DNA Wash Buffer to the column. Centrifuge for min at 13,000 rcf. REPEAT THIS STEP AGAIN

    All of the discarded flow through is collected in a ZR-96 DNA Cleaning waste bottle placed in the waste area next to the molecular hood. Even though the waste collection bottle was made for ZR-96 kit, the buffers that are used in the kit I am using also contains the same buffers and chemicals, hence I emptied the flow-through in the same ZR-96 cleanup waste bottle.

  7. Carefully transfer the spin-column to the final microcentrifuge tubes that can hold your Ultra-pure DNA after the elution step.
  8. Lastly, add 40 μL of Elution Buffer directly to the column matrix and incubate at room temperature. Centrifuge for minute at 13,000 rcf and elute the DNA. The Ultra-pure DNA is now ready to use.

I then plated the Ultra-pure DNA in a 96-well plate with the below Plate map.

Quantification of DNA through Qubit

To make sure that the eluted DNA is of desired concentration, we randomly selected 10 colonies to test their concentration.

Stock solution:

dsDNA Broad Range = 13 X 199 μL= 2587 μL

dsDNA BR reagent= 13 x μL= 13 μL

DNA Standards:

S1= 170.76 μg/mL
S2= 20572.80 μg/mL

Qubit Tube Colony Well Reading Reading 2
  A5 ACR-190 2.96 2.92
2 A12 ACR-180 10.3 10.3
3 B2 ACR-145 5.62 5.66
4 B7 ACR-390 6.54 6.54
5 C4 ACR-360 5.18 5.16
6 C10 ACR-244 7.07 7.00
7 D3 ACR-374 7.39 7.42
8 D8 ACR-231 7.06 7.02
9 E1 ACR-389 8.26 8.24
10 A8 ACR-368 3.76 3.64

Sequencing plate post cleanup

We then sequenced both the forward and reverse for the CRf/CO3r primer set.

Sample information is as follows:

#### CRf forward

Plate 1/2

Sample ID Sample Name Plate Well Template Type PCR Template Volume (µl) H2O Volume (µl) Primer Volume (µl) Primer Name
PP0001 ACR-165 A1 PCR 2 8 2 CRf
PP0002 ACR-193 B1 PCR 2 8 2 CRf
PP0003 ACR-265 C1 PCR 2 8 2 CRf
PP0004 ACR-364 D1 PCR 2 8 2 CRf
PP0005 ACR-389 E1 PCR 2 8 2 CRf
NA Empty F1 empty empty empty empty CRf
NA Empty G1 empty empty empty empty CRf
NA Empty H1 empty empty empty empty CRf
PP0006 ACR-175 A2 PCR 2 8 2 CRf
PP0007 ACR-145 B2 PCR 2 8 2 CRf
PP0008 ACR-256 C2 PCR 2 8 2 CRf
PP0009 ACR-241 D2 PCR 2 8 2 CRf
PP0010 ACR-139 E2 PCR 2 8 2 CRf
NA Empty F2 empty empty empty empty CRf
NA Empty G2 empty empty empty empty CRf
NA Empty H2 empty empty empty empty CRf
PP0011 ACR-186 A3 PCR 2 8 2 CRf
PP0012 ACR-218 B3 PCR 2 8 2 CRf
PP0013 ACR-258 C3 PCR 2 8 2 CRf
PP0014 ACR-374 D3 PCR 2 8 2 CRf
NA Empty E3 empty empty empty empty CRf
NA Empty F3 empty empty empty empty CRf
NA Empty G3 empty empty empty empty CRf
NA Empty H3 empty empty empty empty CRf
PP0015 ACR-187 A4 PCR 2 8 2 CRf
PP0016 ACR-345 B4 PCR 2 8 2 CRf
PP0017 ACR-360 C4 PCR 2 8 2 CRf
PP0018 ACR-393 D4 PCR 2 8 2 CRf
NA Empty E4 empty empty empty empty CRf
NA Empty F4 empty empty empty empty CRf
NA Empty G4 empty empty empty empty CRf
NA Empty H4 empty empty empty empty CRf
PP0019 ACR-190 A5 PCR 2 8 2 CRf
PP0020 ACR-350 B5 PCR 2 8 2 CRf
PP0021 ACR-363 C5 PCR 2 8 2 CRf
PP0022 ACR-237 D5 PCR 2 8 2 CRf
NA Empty E5 empty empty empty empty CRf
NA Empty F5 empty empty empty empty CRf
NA Empty G5 empty empty empty empty CRf
NA Empty H5 empty empty empty empty CRf
PP0023 ACR-351 A6 PCR 2 8 2 CRf
PP0024 ACR-379 B6 PCR 2 8 2 CRf
PP0025 ACR-246 C6 PCR 2 8 2 CRf
PP0026 ACR-176 D6 PCR 2 8 2 CRf
NA Empty E6 empty empty empty empty CRf
NA Empty F6 empty empty empty empty CRf
NA Empty G6 empty empty empty empty CRf
NA Empty H6 empty empty empty empty CRf
PP0027 ACR-347 A7 PCR 2 8 2 CRf
PP0028 ACR-390 B7 PCR 2 8 2 CRf
PP0029 ACR-228 C7 PCR 2 8 2 CRf
PP0030 ACR-225 D7 PCR 2 8 2 CRf
NA Empty E7 empty empty empty empty CRf
NA Empty F7 empty empty empty empty CRf
NA Empty G7 empty empty empty empty CRf
NA Empty H7 empty empty empty empty CRf
PP0031 ACR-368 A8 PCR 2 8 2 CRf
PP0032 ACR-396 B8 PCR 2 8 2 CRf
PP0033 ACR-243 C8 PCR 2 8 2 CRf
PP0034 ACR-231 D8 PCR 2 8 2 CRf
NA Empty E8 empty empty empty empty CRf
NA Empty F8 empty empty empty empty CRf
NA Empty G8 empty empty empty empty CRf
NA Empty H8 empty empty empty empty CRf
PP0035 ACR-140 A9 PCR 2 8 2 CRf
PP0036 ACR-178 B9 PCR 2 8 2 CRf
PP0037 ACR-343 C9 PCR 2 8 2 CRf
PP0038 ACR-229 D9 PCR 2 8 2 CRf
NA Empty E9 empty empty empty empty CRf
NA Empty F9 empty empty empty empty CRf
NA Empty G9 empty empty empty empty CRf
NA Empty H9 empty empty empty empty CRf
PP0039 ACR-150 A10 PCR 2 8 2 CRf
PP0040 ACR-185 B10 PCR 2 8 2 CRf
PP0041 ACR-244 C10 PCR 2 8 2 CRf
PP0042 ACR-220 D10 PCR 2 8 2 CRf
NA Empty E10 empty empty empty empty CRf
NA Empty F10 empty empty empty empty CRf
NA Empty G10 empty empty empty empty CRf
NA Epty H10 empty empty empty empty CRf
PP0043 ACR-173 A11 PCR 2 8 2 CRf
PP0044 ACR-210 B11 PCR 2 8 2 CRf
PP0045 ACR-213 C11 PCR 2 8 2 CRf
PP0046 ACR-231 D11 PCR 2 8 2 CRf
NA Empty E11 empty empty empty empty CRf
NA Empty F11 empty empty empty empty CRf
NA Empty G11 empty empty empty empty CRf
NA Empty H11 empty empty empty empty CRf
PP0047 ACR-180 A12 PCR 2 8 2 CRf
PP0048 ACR-51 B12 PCR 2 8 2 CRf
PP0049 ACR-234 C12 PCR 2 8 2 CRf
PP0050 ACR-267 D12 PCR 2 8 2 CRf
NA Empty E12 empty empty empty empty CRf
NA Empty F12 empty empty empty empty CRf
NA Empty G12 empty empty empty empty CRf
NA Empty H12 empty empty empty empty CRf

#### CO3r reverse

Plate 2/2

Sample ID Sample Name Plate Well Template Type PCR Template Volume (µl) H2O Volume (µl) Primer Volume (µl) Primer Name
PP0051 ACR-165 A1 PCR 2 8 2 C03r
PP0052 ACR-193 B1 PCR 2 8 2 C03r
PP0053 ACR-265 C1 PCR 2 8 2 C03r
PP0054 ACR-364 D1 PCR 2 8 2 C03r
PP0055 ACR-389 E1 PCR 2 8 2 C03r
NA Empty F1 empty empty empty empty C03r
NA Empty G1 empty empty empty empty C03r
NA Empty H1 empty empty empty empty C03r
PP0056 ACR-175 A2 PCR 2 8 2 C03r
PP0057 ACR-145 B2 PCR 2 8 2 C03r
PP0058 ACR-256 C2 PCR 2 8 2 C03r
PP0059 ACR-241 D2 PCR 2 8 2 C03r
PP0060 ACR-139 E2 PCR 2 8 2 C03r
NA Empty F2 empty empty empty empty C03r
NA Empty G2 empty empty empty empty C03r
NA Empty H2 empty empty empty empty C03r
PP0061 ACR-186 A3 PCR 2 8 2 C03r
PP0062 ACR-218 B3 PCR 2 8 2 C03r
PP0063 ACR-258 C3 PCR 2 8 2 C03r
PP0064 ACR-374 D3 PCR 2 8 2 C03r
NA Empty E3 empty empty empty empty C03r
NA Empty F3 empty empty empty empty C03r
NA Empty G3 empty empty empty empty C03r
NA Empty H3 empty empty empty empty C03r
PP0065 ACR-187 A4 PCR 2 8 2 C03r
PP0066 ACR-345 B4 PCR 2 8 2 C03r
PP0067 ACR-360 C4 PCR 2 8 2 C03r
PP0068 ACR-393 D4 PCR 2 8 2 C03r
NA Empty E4 empty empty empty empty C03r
NA Empty F4 empty empty empty empty C03r
NA Empty G4 empty empty empty empty C03r
NA Empty H4 empty empty empty empty C03r
PP0069 ACR-190 A5 PCR 2 8 2 C03r
PP0070 ACR-350 B5 PCR 2 8 2 C03r
PP0071 ACR-363 C5 PCR 2 8 2 C03r
PP0072 ACR-237 D5 PCR 2 8 2 C03r
NA Empty E5 empty empty empty empty C03r
NA Empty F5 empty empty empty empty C03r
NA Empty G5 empty empty empty empty C03r
NA Empty H5 empty empty empty empty C03r
PP0073 ACR-351 A6 PCR 2 8 2 C03r
PP0074 ACR-379 B6 PCR 2 8 2 C03r
PP0075 ACR-246 C6 PCR 2 8 2 C03r
PP0076 ACR-176 D6 PCR 2 8 2 C03r
NA Empty E6 empty empty empty empty C03r
NA Empty F6 empty empty empty empty C03r
NA Empty G6 empty empty empty empty C03r
NA Empty H6 empty empty empty empty C03r
PP0077 ACR-347 A7 PCR 2 8 2 C03r
PP0078 ACR-390 B7 PCR 2 8 2 C03r
PP0079 ACR-228 C7 PCR 2 8 2 C03r
PP0080 ACR-225 D7 PCR 2 8 2 C03r
NA Empty E7 empty empty empty empty C03r
NA Empty F7 empty empty empty empty C03r
NA Empty G7 empty empty empty empty C03r
NA Empty H7 empty empty empty empty C03r
PP0081 ACR-368 A8 PCR 2 8 2 C03r
PP0082 ACR-396 B8 PCR 2 8 2 C03r
PP0083 ACR-243 C8 PCR 2 8 2 C03r
PP0084 ACR-231 D8 PCR 2 8 2 C03r
NA Empty E8 empty empty empty empty C03r
NA Empty F8 empty empty empty empty C03r
NA Empty G8 empty empty empty empty C03r
NA Empty H8 empty empty empty empty C03r
PP0085 ACR-140 A9 PCR 2 8 2 C03r
PP0086 ACR-178 B9 PCR 2 8 2 C03r
PP0087 ACR-343 C9 PCR 2 8 2 C03r
PP0088 ACR-229 D9 PCR 2 8 2 C03r
NA Empty E9 empty empty empty empty C03r
NA Empty F9 empty empty empty empty C03r
NA Empty G9 empty empty empty empty C03r
NA Empty H9 empty empty empty empty C03r
PP0089 ACR-150 A10 PCR 2 8 2 C03r
PP0090 ACR-185 B10 PCR 2 8 2 C03r
PP0091 ACR-244 C10 PCR 2 8 2 C03r
PP0092 ACR-220 D10 PCR 2 8 2 C03r
NA Empty E10 empty empty empty empty C03r
NA Empty F10 empty empty empty empty C03r
NA Empty G10 empty empty empty empty C03r
NA Empty H10 empty empty empty empty C03r
PP0093 ACR-173 A11 PCR 2 8 2 C03r
PP0094 ACR-210 B11 PCR 2 8 2 C03r
PP0095 ACR-213 C11 PCR 2 8 2 C03r
PP0096 ACR-231 D11 PCR 2 8 2 C03r
NA Empty E11 empty empty empty empty C03r
NA Empty F11 empty empty empty empty C03r
NA Empty G11 empty empty empty empty C03r
NA Empty H11 empty empty empty empty C03r
PP0097 ACR-180 A12 PCR 2 8 2 C03r
PP0098 ACR-51 B12 PCR 2 8 2 C03r
PP0099 ACR-234 C12 PCR 2 8 2 C03r
PP00100 ACR-267 D12 PCR 2 8 2 C03r
NA Empty E12 empty empty empty empty C03r
NA Empty F12 empty empty empty empty C03r
NA Empty G12 empty empty empty empty C03r
NA Empty H12 empty empty empty empty C03r

20250915

The sequencing plates were sent off for sequencing to Janet Atoyan at the RI Genomics & Sequencing Center in the loading dock freezer of the pharmacy building.

Next Steps

We will analyze sequences to determine species ID and then decide how to proceed further.

Written on September 5, 2025