PocID Relative abundance molecular species ID.

Relative abundance Pocillopora spp. around the island of Mo’orea. Species identification via mtORF-PocHistone and RFLP.

Context:

Our study is based on population genetics of Pocillopora sp. on the Polynesian islands based on their often misleading and incorrect morphology. We are therefore trying to understand population dynamics by Haplotype so that we can correlate our transect data with our term tolerance data from the laboratory (see NoteBook TPC Haplotype).

gDNA extraction with “Quick-DNA 96 Kit / Cat No: D3012

Protocol:

  1. Reagent preparation: Add 260ul or 1,040ul Proteinase K storage Buffer to reconstitute the lyophilized Proteinase K, 5mg (D3001-2-5) or 20mg (D3001-2-20), respectively (final concentration of 20mg/ml). Vortex to dissolve.

  2. gDNA extraction in DNA/RNA shield

    • Add 10ul PK Digestion buffer + 5ul of Proteinase K.
      For 2 plate in a time –> 2.0ml of PK Digestion Buffer + 1.0ml ProtK

    • Add 400ul og Genomic Lysis Buffer to 100ul of teh sample/shield mixture.
      For 2 plate in a time –> 400ul x 192 samples = 76.8ml

    • Mix completely by vortexing (with pipette in the plate) 4-5 times then let stand 10 minutes

    • Transfer the mixture to the wells of a Silicon-A Plate on a Collection Plate. Centrifuge at 2.500xg for 5 minutes.

    • Remove the waste in the colelction plate and move to the next step.

    • Add 200ul of DNA Pre-Wash Buffer to each well and. centrifuge at 2,500xg for 5 nminutes,
      For 2 plate in a time –> 200ul x 192 samples = 38.4ml.

    • Remove the waste in the colelction plate and move to the next step.

    • Add 300ul g-DNA Wash Buffer to each well and centrifuge at 2,500xg for 5 minutes.
      For 2 plate in a time –> 300ul x 192 samples = 57.6ml.

    • Transfer the Silicon-A Plate onto an Elution plate. Add 50ul DNA Elution Buffer (need to be heat at 70˚C) to each well and incubate during 2 minutes then centrifuge at 2,500xg for 5 minutes. For 2 plate in a time –> 50ul x 192 samples = 9.6ml.

    • Keep the Silicon-A Plate in the fridge (4˚C).

    • Cover the Elution Plate and keep it at -20˚C.

    • Nano drop 3-4 samples on each plate to control de DNA concentration.

Nano drop result :

20240919 Plate 16 and 17.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
17 1398 A1 17 1.72 0.85
17 1390 B1 1.1 1.6 0.16
16 1407 H12 1.5 2.75 0.56
16 2220 A1 .- 0,8 0.84 .-0.06
16 2194 B1 .-0.9 1.16 .-0.25

After checking we figure it out that we didin’t add Elution Buffer but g-DNA Wash buffer. Following that we tooked plates out of the freezer and added 50ul of Elution Buffer Eluted in 50ul of Elution Buffer and mesured on nanodrop.

Plate Sample Well ng/ul A260/280 A260/230
16 2220 A1 3.4 1.97 0.89
16 2194 B1 20.8 1.88 1.83
16 2189 C1 13.6 1.9 1.96
17 1398 A1 10 1.82 1.01
17 1390 B1 14.9 1.86 2.32
17 2163 C1 12.1 1.91 1.31

20240920 Plate 18 and 19.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.1 .-0.14 0.18
18 2418 A1 15.6 2.11 2.3
18 2350 B1 23.8 2.05 0.33
18 2421 C1 31.2 2.03 1.64
19 1448 A1 16.9 2.1 2.16
19 1442 B1 12 2.14 2.22
19 1418 C1 4.1 2.83 0.56

20240920 Plate 20 and 21.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.1 .-1.64 0.58
20 1424 A1 14.3 2.08 2.05
20 2463 B1 33.2 1.97 1.46
20 3314 C1 28.1 1.94 1.58
21 2137 A1 5.6 2.11 1.04
21 2152 B1 10.4 1.97 2.22
21 3113 C1 24.4 1.97 2.02

Probability of mixing buble surface between 12C and 12D on plate 20.

20240920 Plate 22 and 23.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.8 .-12.88 2.26
22 2136 A1 18.1 1.94 1.48
22 3231 B1 39 1.95 1.89
22 3291 C1 22.8 1.98 1.64
23 3496 A1 25.8 2 2.46
23 3620 B1 19 1.98 1.65
23 2916 C1 37.6 1.93 2.42

20240921 Plate 24 and 25.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0 .-0.01 0.12
24 3513 A12 61.8 1.96 2.33
24 3501 B12 34 2.02 1.75
24 3674 C12 30.8 2.01 1.76
25 2752 F12 21.8 2.05 2.45
25 2781 G12 20.1 2.07 2.26
25 2768 H12 43.4 2.01 2.27

20240922 Plate 26 and 27.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.2 2.78 0.43
26 2715 A1 39 1.93 1.72
26 2708 B1 35.4 1.9 0.93
26 2845 C1 32 1.95 1.55
27 3445 A12 42.9 1.92 0.84
27 3679 B12 8.9 2.11 1.83
27 2694 C12 15.9 1.98 1.4

20240922 Plate 28 and 29.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x .-0.4 1.3 .-5.44
28 2386 F1 33.5 1.91 1.94
28 2382 G1 38 1.93 1.9
28 2361 H1 35.9 1.92 1.88
29 2996 A1 34.1 1.95 1.6
29 2855 B1 42.1 1.9 1.09
29 2605 C1 35.2 1.95 2.14

20240922 Plate 30 and 31.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x .-0.1 0.27 .-0.95
31 3385 A12 31.9 1.93 2.05
31 3364 B12 29.1 1.94 2.02
31 3431 C12 10.5 1.83 0.53
30 2665 F12 35.8 1.91 2.18
30 2991 G12 28.5 1.94 1.63
30 2500 H12 47.9 1.9 1.81

20240923 Plate 32 and 33.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x .-0.1 0.18 .-0.18
32 430 A1 35.6 1.96 2.1
32 2085 B1 12.6 2.1 1.62
32 1975 C1 36.8 1.99 1.87
33 2004 A12 34.4 1.95 1.38
33 1914 B12 29.6 2 1.71
33 1779 C12 19.9 2.04 2.08

20240923 Plate 34 and 35.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.2 .-0.45 0.88
34 1530 A1 19.7 1.97 1.68
34 1659 B1 24.7 2.02 1.87
34 1567 C1 22.6 2.02 2.07
35 1527 F12 46.5 1.92 2.11
35 1928 G12 31.1 1.94 1.63
35 1985 H12 38.9 1.95 2.19

20240924 Plate 36 and 37.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.1 .-0.09 .-0.32
36 1959 A1 62.1 1.93 2.37
36 1963 B1 16.5 2.03 2.28
36 2025 C1 23.5 1.99 2.64
37 931 A10 54.4 1.96 2.13
37 1218 A11 32.7 1.93 2.7
37 1075 A12 47.8 1.93 1.81

20240925 Plate 38 and 39.

See plate number for each samples here.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.3 .-0.30 .-1.36
38 814 A1 31 1.99 2.13
38 892 A2 45.1 1.97 1.8
38 799 A3 24.4 2.04 1.16

Gel process control of DNA quality

Protocol:

During the process we used differentes size of gel. For this raison we have list bellow all the different way to make the gel at 1.5%.

Small size of gel:
  • 1.12g of Agarose
  • 75ml of 1x TAE Buffer
  • 1µl of gelgreen.
Medium size of gel:
  • 1.50g of Agarose
  • 100ml of 1x TAE Buffer
  • 1µl of gelgreen.
Large size of gel:
  • 2.25 of Agarose
  • 150ml of 1x TAE Buffer
  • 1µl of gelgreen.

On each gel add 4µl of lader on one side of the gel. On each gel add 4µl of samples in each well. Run the gel at 100V during 35 minutes.

Layer use for mtORF

QuickLoadpurple_mtORF.JPG

Gel pictures

20240411 plate002 gel 001 mtORF after extraction:

P02g1.JPG

20240411 plate002 gel 002 mtORF after extraction:

P02g2.JPG

20240411 plate002 gel 003 mtORF after extraction:

P02g3.JPG

20250226 plate003 gel 001 mtORF after extraction:

P03g1.JPG

20250226 plate003 gel 003 mtORF after extraction:

P03g3.JPG

20250226 plate004 gel 001 mtORF after extraction:

P04g1.JPG

20250226 plate004 gel 002 mtORF after extraction:

P04g2.JPG

20250226 plate004 gel 003 mtORF after extraction:

P04g3.JPG

20250226 plate005 gel 001 mtORF after extraction:

P05g1.JPG

20250226 plate005 gel 002 mtORF after extraction:

P05g2.JPG

20250226 plate005 gel 003 mtORF after extraction:

P05g3.JPG

20250226 plate006 gel 001 mtORF after extraction:

P06g1.JPG

20250226 plate006 gel 002 mtORF after extraction:

P06g2.JPG

20250226 plate006 gel 003 mtORF after extraction:

P06g3.JPG

20250226 plate007 gel 001 mtORF after extraction:

P07g1.JPG

20250226 plate007 gel 002 mtORF after extraction:

P07g2.JPG

20250226 plate007 gel 003 mtORF after extraction:

P07g3.JPG

20250227 plate008 gel 001 mtORF after extraction:

P08g1.JPG

20250227 plate008 gel 002 mtORF after extraction:

P08g2.JPG

20250227 plate008 gel 003 mtORF after extraction:

P08g3.JPG

20250227 plate009 gel 001 mtORF after extraction:

P09g1.JPG

20250227 plate009 gel 002 mtORF after extraction:

P09g2.JPG

20250227 plate009 gel 003 mtORF after extraction:

P09g3.JPG

20241005 plate012 mtORF after extraction:

P12.JPG

20241005 plate013 mtORF after extraction:

P13.JPG

20241005 plate014 mtORF after extraction:

P14.JPG

20241005 plate015 mtORF after extraction:

P15.JPG

20241005 plate016 mtORF after extraction:

P16.JPG

2024xxxx plate017 mtORF after extraction:

P17.JPG

20241005 plate018 mtORF after extraction:

P18.JPG

2024xxxx plate019 mtORF after extraction:

P19.JPG

2024xxxx plate020 gel 001 mtORF after extraction:

P20g1.JPG

2024xxxx plate020 gel 002 mtORF after extraction:

P20g2.JPG

2024xxxx plate020 gel 003 mtORF after extraction:

P20g3.JPG

2024xxxx plate021 gel 001 mtORF after extraction:

P21g1.JPG

2024xxxx plate021 gel 002 mtORF after extraction:

P21g2.JPG

2024xxxx plate021 gel 003 mtORF after extraction:

P21g3.JPG

20240928 plate022 gel 001 mtORF after extraction:

P22g1.JPG

20240928 plate022 gel 002 mtORF after extraction:

P22g2.JPG

20240928 plate022 gel 003 mtORF after extraction:

P22g3.JPG

20240928 plate023 gel 001 mtORF after extraction:

P23g1.JPG

20240928 plate023 gel 002 mtORF after extraction:

P23g2.JPG

20240928 plate023 gel 003 mtORF after extraction:

P23g3.JPG

20240930 plate024 gel 001 mtORF after extraction:

P24g1.JPG

20240930 plate024 gel 002 mtORF after extraction:

P24g2.JPG

20240930 plate024 gel 003 mtORF after extraction:

P24g3.JPG

20240930 plate025 gel 001 mtORF after extraction:

P25g1.JPG

20240930 plate025 gel 002 mtORF after extraction:

P25g2.JPG

20240930 plate025 gel 003 mtORF after extraction:

P25g3.JPG

20240930 plate026 gel 001 mtORF after extraction:

P26g1.JPG

20240930 plate026 gel 002 mtORF after extraction:

P26g2.JPG

20240930 plate026 gel 003 mtORF after extraction:

P26g3.JPG

20240930 plate027 gel 001 mtORF after extraction:

P27g1.JPG

20240930 plate027 gel 002 mtORF after extraction:

P27g2.JPG

20240930 plate027 gel 003 mtORF after extraction:

P27g3.JPG

20241001 plate028 gel 001 mtORF after extraction:

P28g1.JPG

20241001 plate028 gel 002 mtORF after extraction:

P28g2.JPG

20241001 plate028 gel 003 mtORF after extraction:

P28g3.JPG

20241001 plate029 gel 001 mtORF after extraction:

P29g1.JPG

20241001 plate029 gel 002 mtORF after extraction:

P29g2.JPG

20241001 plate029 gel 003 mtORF after extraction:

P29g3.JPG

20241001 plate030 gel 001 mtORF after extraction:

P30g1.JPG

20241001 plate030 gel 002 mtORF after extraction:

P30g2.JPG

20241001 plate030 gel 003 mtORF after extraction:

P30g3.JPG

20241001 plate031 gel 001 mtORF after extraction:

P31g1.JPG

20241001 plate031 gel 002 mtORF after extraction:

P31g2.JPG

20241001 plate032 gel 001 mtORF after extraction:

P32g3.JPG

20241001 plate032 gel 002 mtORF after extraction:

P32g2.JPG

20241002 plate033 gel 001 mtORF after extraction:

P33g1.JPG

20241002 plate033 gel 002 mtORF after extraction:

P33g2.JPG

20241002 plate033 gel 003 mtORF after extraction:

P33g3.JPG

20241002 plate034 gel 001 mtORF after extraction:

P34g1.JPG

20241002 plate034 gel 002 mtORF after extraction:

P34g2.JPG

20241002 plate034 gel 003 mtORF after extraction:

P34g3.JPG

20241002 plate035 gel 001 mtORF after extraction:

P35g1.JPG

20241002 plate035 gel 002 mtORF after extraction:

P35g2.JPG

20241002 plate036 gel 001 mtORF after extraction:

P36g1.JPG

20241002 plate036 gel 002 mtORF after extraction:

P36g2.JPG

20241002 plate036 gel 003 mtORF after extraction:

P36g3.JPG

20241002 plate037 gel 001 mtORF after extraction:

P37g1.JPG

20241002 plate037 gel 002 mtORF after extraction:

P37g2.JPG

20241002 plate037 gel 003 mtORF after extraction:

P37g3.JPG

20241002 plate038 gel 001 mtORF after extraction:

P38g1.JPG

20241002 plate038 gel 002 mtORF after extraction:

P38g2.JPG

20241002 plate038 gel 003 mtORF after extraction:

P38g3.JPG

20241002 plate039 gel 001 mtORF after extraction:

P39g1.JPG

20241002 plate039 gel 002 mtORF after extraction:

P39g2.JPG

ZR 96 DNA Clean-up Kit Cat No D4017 D4018

Protocol:

  1. Reagent preparation: Add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA Wash Buffer concentrate. Add 192 ml 100% ethanol (208 ml 95% ethanol) to the 48ml DNA Wash Buffer concentrate.

  2. Sample processing:

  • Add 100 µl DNA Binding Buffer to PCR sample, Mix, transfer to cullum plate, then add any remaning sample (20µl).

  • Centrifuge at 3000g for 5 minutes

  • Add 300 µl Wash buffer to each well

  • Centrifuge at 3000g for 5 minutes

  • Add 300 µl at Wash buffer

  • Centrifuge at 3000g for 5 minutes

  • Add 40µl nuc free water to each well

  • Transfer silicon plate into an evolution plate

  • Centrifuge 3000g for 3 minutes

  • Done! Store in freezer

Sequence process at URI

PocHistone and RFLP for species identify as Haplotype 1a (P. grandis - P. meandrina):

Protocol:

The protocol used comes from Hollie Putnam’s LabNotebook and is available here

Layer use for PocHistone RFLP

QuickLoadpurple_RFLP.JPG

Gel pictures

20240303 PocHistone RFLP 001 gel 1:

pocHistone001g1.JPG

20240303 PocHistone RFLP 001 gel 2:

pocHistone001g2.JPG

20240303 PocHistone RFLP 001 gel 3:

pocHistone001g3.JPG

2024xxxx PocHistone RFLP 002 gel 1:

pocHistone002g1.JPG

2024xxxx PocHistone RFLP 002 gel 2:

pocHistone002g2.JPG

2024xxxx PocHistone RFLP 003 gel 1:

pocHistone003g1.JPG

2024xxxx PocHistone RFLP 003 gel 2:

pocHistone003g2.JPG

2024xxxx PocHistone RFLP 003 gel 3:

pocHistone003g3.JPG

Resume of PocHistone RFLP Haplotype 1a gel:

P. meandrina P. grandis Did not work
433 456 549
675 601 3536
451 607 3872
431 550 3919
652 458 3729
602 410 3751
442 559 3789
461 422 -
441 557 -
418 726 -
597 559 -
440 683 -
429 689 -
603 670 -
605 3559 -
439 3549 -
400 667 -
427 3735 -
573 3613 -
575 3536 -
426 1447 -
606 3531 -
556 3621 -
528 3732 -
479 3535 -
448 3549 -
412 3559 -
424 3613 -
411 3709 -
449 3787 -
434 3793 -
592 3949 -
445 3680 -
604 3641 -
614 3692 -
726 3735 -
3617 3736 -
2550 3879 -
676 3757 -
3724 3777 -
3707 3782 -
668 3807 -
3532 3812 -
596 3895 -
685 - -
680 - -
725 - -
3723 - -
723 - -
3615 - -
3726 - -
721 - -
3710 - -
3686 - -
3592 - -
3629 - -
535 - -
703 - -
3552 - -
3627 - -
3602 - -
702 - -
666 - -
3610 - -
684 - -
2189 - -
2185 - -
2205 - -
2186 - -
2213 - -
2192 - -
2184 - -
1423 - -
3537 - -
3553 - -
3558 - -
3622 - -
3626 - -
3628 - -
3706 - -
3713 - -
3719 - -
3728 - -
3732 - -
3685 - -
3703 - -
3759 - -
3769 - -
3843 - -
3871 - -
3881 - -
3522 - -
3550 - -
3552 - -
3592 - -
3602 - -
3610 - -
3615 - -
3617 - -
3627 - -
3629 - -
3707 - -
3710 - -
3724 - -
3726 - -
3544 - -
3545 - -
3548 - -
3595 - -
3600 - -
3624 - -
3633 - -
3635 - -
3643 - -
3711 - -
3720 - -
3691 - -
3696 - -
3699 - -
3733 - -
3734 - -
3742 - -
3747 - -
3751 - -
3755 - -
3758 - -
3764 - -
3770 - -
3818 - -
3839 - -
3850 - -
3856 - -
3860 - -
3862 - -
3874 - -
3884 - -
3808 - -
3809 - -
3818 - -
3827 - -
3890 - -
3903 - -
3904 - -
3908 - -
3914 - -
3915 - -
3918 - -
3942 - -
3591 - -
3601 - -
3609 - -
3715 - -
3686 - -
3688 - -
3698 - -
3700 - -
3701 - -
3737 - -
3744 - -
3745 - -
3746 - -
3750 - -
3761 - -
3765 - -
3767 - -
3772 - -
3833 - -
3836 - -
3846 - -
3848 - -
3852 - -
3863 - -
3864 - -
3865 - -
3869 - -
3873 - -
3877 - -
3885 - -
3739 - -
3741 - -
3743 - -
3766 - -
3771 - -
3834 - -
3835 - -
3837 - -
3841 - -
3847 - -
3854 - -
3855 - -
3859 - -
3866 - -
3887 - -
3889 - -
3774 - -
3776 - -
3783 - -
3784 - -
3785 - -
3786 - -
3788 - -
3795 - -
3797 - -
3798 - -
3800 - -
3803 - -
3806 - -
3811 - -
3816 - -
3817 - -
3823 - -
3824 - -
3829 - -
3830 - -
3897 - -
3912 - -
3913 - -
3917 - -
3934 - -
3935 - -
3937 - -
3934 - -
3935 - -
3937 - -
3938 - -
3943 - -
3945 - -
3789 - -
3804 - -
Written on October 9, 2024