Testing Chelex-extracted DNA in comparison to direct PCR of tissue for Pocillopora ID in the field

Equipment and Reagents

• Bento Lab Bento Lab thermal cycler, gel, centrifuge
• BSA Thermo Scientific™ Pierce™ Bovine Serum Albumin Standard Ampules, 2mg/ml, Catalog #PI23209
• F primer FatP6.1 200µM Stock IDT)
• R primer RORF 200µM Stock IDT
• Gel ladder GeneRuler 1kb plus DNA Ladder (Thermo FIsher Catalog SM1333)
• Master Mix FERK1071 DREAMTAQ Master Mix
• Direct Master Mix F170S PHIRE TISSUE DIRECT PCR Master Mix
• Gel Stain Biotium GelGreen Nucleic Acid Gel Stain, 10,000X in Water Fisher Cat NC9728313

Prepare Reagents

• diluted BSA from 2mg/ml to 0.25mg/ml in Nuclease Free water
• F primer FastP6.1 (Added 50µl of stock 2mg/ml plus 950µl of Nuclease Free water and made 3 aliquots of ~320µl each and stored in reagent box at -20)
• R primer RORF (Added 50µl of stock 2mg/ml plus 950µl of Nuclease Free water and made 3 aliquots of ~320µl each and stored in reagent box at -20)

Chelex DNA Extraction was completed on April 9th 2022 and DNA stored at -20°C

TESTING mtORF AMPLIFICATION OF CHELEX EXTRACTED DNA FROM 4/9/22

Dream Taq PCR Master Mix (2x)

Master Mix:

Reagent	1Rxn µl	10 Rxn µl
Phire Taq Mix	7.5	75
BSA (0.25mg/ml)	0.14	1.4
F primer FastP6.1 (10µM)	0.195	1.95
R primer RORF	0.195	1.95
DNA	1.5	NA
H2O	5.47	54.7
Total volume	15	150

Thermal Cycling Conditions

• [95°C 60 secondes] 1 cycle
• [95°C 30 sec,53°C 30 sec, 72°C 70 sec] 40 cycles
• [72°C 5 minutes] 1 cycle
• [15°C infinity]

Sample

1: H1 Hollie 1 chelex extraction of P. acuta 4/9/22
2: H2 Hollie 2 chelex extraction of P. acuta 4/9/22
3: H3 Hollie negative extraction control 4/9/22
4: P1 Pierrick 1 chelex extraction of P. acuta 4/9/22
5: P2 Pierrick 2 chelex extraction of P. acuta 4/9/22
6: P3 Pierrick negative extraction control 4/9/22
7: H20 Negative PCR Control
8: 253= Pocillopora gDNA from E5 Terpis

Gel protocol

prepared a 50ml gel using 50ml of 1xTAE + 0.5g Agarose, melted gel for ~90 sec in the microwave and let cool and added 1µl Gelgreen

Ran the gel in 0.25X TAE to reduce heating in the Bento Lab, but had to move the gel to on of the OWl rigs in order to start the next PCR in the Bento Lab.

Gel Results

3 of the 4 coral samples extracted with Chelex amplified the desired band with the mtORF primers along with the positive control

TESTING mtORF AMPLIFICATION DIRECT FROM CORAL SAMPLES

Phire Taq Mix Direct PCR

Phire Master Mix:

Reagent	1Rxn µl	7 Rxn µl
Phire Taq Mix	7.5	52.5
BSA (0.25mg/ml)	NA	NA
F primer FastP6.1 (10µM)	0.195	1.365
R primer RORF (10µM)	0.195	1.365
DNA	Direct Clipping	Direct Clipping
H2O	7.11	49.77
Total volume	15	105

Aliquot 13.5µl of master mix into each tube

Phire Thermal Cycling Conditions

• [98°C 5 minutes] 1 cycle
• [98°C 10 sec, 58°C 10 sec, 72°C 20 sec] 40 cycles
• 72°C 1 minute 1 cycle
• 15°C infinity
• The protocol calls for 5 second steps, but the Bento Lab shortest step time is 10seconds

Phire Samples

scraped samples from the coral plug P. acuta before scraping all 3 samples (left) and after (right)

1: Cl= Clip1 - largest piece ~2mm x 2mm
2: C2= Clip2 - piece 1mm x 1mm
3: C3= Clip3 - piece 0.5mm x 0.5mm
4: Neg= H2O
5: H1= Chelex 1
6: 253= gDNA 253 Poc from E5 Terpis
7: C4= Clip4 - Scraped w/pipette tip only
8: P2 from above run again

Phire Gel protocol

prepared a 50ml gel using 50ml of 1xTAE + 0.5g Agarose, melted gel for ~90 sec in the microwave and let cool and added 1µl Gelgreen

Ran the gel in 0.25X TAE to reduce heating in the Bento Lab, but gel melting was still visible.

Phire Gel Results

The clippings for the coral inhibited the PCR, the only sample that worked for the direct from tissue PCR was the “Clip 4” which was only coral tissue rubbed on the side of the plug with the pipette tip. The chelex sample H1 and the gDNA E5 sample 253 also worked.

It seems less is more in this case, so this bodes well for taking swabs of the coral DNA from recruitment plates.