Poc ID via mtORF POC RAPID Plate 008 Plate 009
gDNA extraction for Pocillopora species ID COTS POC RAPID Plate008 and Plate009
Equipment and Reagents
- Sample Preservation and Lysis Zymo Research DNA/RNA Shield R1100-250
- DNA Extraction 96 Well Plate Zymo Research Quick-DNA™ 96 D3010
- Proteinase K Zymo Proteinase K with Storage BufferZymo D3001-2-20
- PK Digestion Buffer Zymo PK Digestion Buffer R1200-1-20
- Eppendorf Deepwell Plate 96/2000 µL plates Eppendorf Cat # 951033502
09 May 2024
Plate preparation of Plate 008 and Plate 009
Moved 100µl of sample liquid (lysed sample in Zymo RNA/DNA Shield) to deep well 96 eppendorf plate for 96 samples x 2 plates = 192 samples.
Samples
DNA Extraction
DNA was extracted withZymo Research Quick-DNA™ 96 Kit D3010
Used Page 10 Proteinase K Digestion with DNA/RNA Shield
Used Eppendorf Deepwell Plate 96/2000 µL plates Catalog No. 951033502 for reagent and sample mixing prior to adding to Zymo silicon column plate
The samples were extracted according to the manufacturer’s instructions for the Quick DNA 96 Kit for samples stored in DNA/RNA Shield including the addition of Proteinase K (20mg ml-1).
Added 100µl of sample + 10µl of PK Digestion Buffer + 5µl of ProteinaseK. Pipetted to mix and incubated for 5 min at room temp.
- 4 volumes of Zymo Kit Genomic Lysis Buffer was added for each volume of the sample digestion (e.g., add 400µl of Genomic Lysis Buffer to 100µl of sample digestion). Samples were mixed via pipetting and the mixtures were incubated for 5 minutes at room temperature. Then samples were moved to the silicon-A plate columns. I added 400µl and then 5 minute spin at 2500 rcf.
The waste was removed from the collection plate after the first centrifugation step.
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Next 200µl of DNA Pre-wash buffer was added to each well and then the silicon-A was spun for 5 minute at 2500 rcf
- used sterile 25 or 50ml plastic troughs and multi-channel pipettes
- 192 samples x 200µl = 38.4ml
- Next 300µl of g-DNA wash buffer was added to each well and then the silicon-A was spun for 5 minute at 2500 rcf
- used sterile 25 or 50ml plastic troughs and multi-channel pipettes
- 192 samples x 300µl = 57.6ml
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Moved the silicon-A plate from the collection plate to an Elution plate
- DNA was then eluted in 50µl of kit Elution Buffer warmed to 70°C and standing on the columns for 3-5 minutes.
- used sterile 25 or 50ml plastic troughs and multi-channel pipettes
- 192 samples x 50µl = 9.6ml
Quantification of gDNA
Used Lane lab nanodrop and kit elution buffer as blank to quantify the DNA. Measured 1.5µl of sample.
| Sample ID | Well | Project | ng/µl | A260/280 | A260/230 | gDNA Plate |
|---|---|---|---|---|---|---|
| 1052 | A1 | POC COTS RAPID RA | 18.4 | 1.94 | 2.34 | Plate008 |
| 1142 | B1 | POC COTS RAPID RA | 22.7 | 1.97 | 1.59 | Plate008 |
| 1123 | C1 | POC COTS RAPID RA | 22.9 | 1.95 | 2.25 | Plate008 |
| 933 | D1 | POC COTS RAPID RA | 21.2 | 1.98 | 1.80 | Plate008 |
| 973 | E1 | POC COTS RAPID RA | 19.8 | 1.97 | 1.90 | Plate008 |
| 1007 | F1 | POC COTS RAPID RA | 24.5 | 2.00 | 2.18 | Plate008 |
| 1015 | G1 | POC COTS RAPID RA | 4.1 | 1.95 | 2.65 | Plate008 |
| 907 | H1 | POC COTS RAPID RA | 29.2 | 1.95 | 2.18 | Plate008 |
| 1219 | A1 | POC COTS RAPID RA | 29.9 | 1.92 | 2.45 | Plate009 |
| 1204 | B1 | POC COTS RAPID RA | 25.6 | 1.95 | 1.84 | Plate009 |
| 1194 | C1 | POC COTS RAPID RA | 8.8 | 1.93 | 0.78 | Plate009 |
| 1150 | D1 | POC COTS RAPID RA | 30.8 | 1.87 | 2.15 | Plate009 |
| 1191 | E1 | POC COTS RAPID RA | 17.8 | 2.01 | 2.14 | Plate009 |
| 1333 | F1 | POC COTS RAPID RA | 1.9 | 3.96 | 8.02 | Plate009 |
| 1339 | G1 | POC COTS RAPID RA | 20.5 | 2.00 | 2.10 | Plate009 |
| 468 | H1 | POC COTS RAPID RA | 19.2 | 2.00 | 1.56 | Plate009 |
All tested samples had DNA and can move forward for PCR.
gDNA was stored at -20°C in the elution plate covered with a foil seal.