2TRNL Mule Deer

Samples arrived 05-05-2022. Samples checked in against list 7-13-2022.

Plates 1-3 have been extracted, normalized, PCRed, quantified and normalized by 7-15-22.

16S for 2TRNL1 was re-PCRed with G4 and H4 because there was low amplification with A5 and B5.

Extractions

Transfer fecal material to labeled bead tubes. Weigh first few samples to approximate amount of material ~200mg

Freeze dry samples

Extract using MN Nucleospin 96 Stool kits following spin speeds from MN Nucleospin 96 Soil kits

Transfer DNAs to plate

Transfer 30 ul as working aliquot

Add 4 samples of 5 ng/ul Mock Community Only and 4 samples of half mock community half DNA from a single plant sample DNA to column 9(first empty column) of 2TRNL8

Add ISD and coligos

Normalize

PCR MasterMix (28 Plates)

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	2690	8,070
0.45	10M dNTPs	2690	1211
0.3	Kapa HiFi HotStart DNA Pol	2690	807
7.25	HPLC H2O	2690	19,502
11	Total Volume	2690	29,590

Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.

Plate	TRNL Primers	16S Primers

Plate	TRNL Primers	16S Primers
2TRNL1	TRNL01	16S0A5
2TRNL1	TRNL02	16S0B5
2TRNL2	TRNL03	16S0C5
2TRNL2	TRNL04	16S0D5
2TRNL3	TRNL05	16S0E5
2TRNL3	TRNL06	16S0F5
2TRNL4	TRNL07	16S0G5
2TRNL4	TRNL08	16S0H5
2TRNL5	TRNL09	16S0A6
2TRNL5	TRNL10	16S0B6
2TRNL6	TRNL11	16S0C6
2TRNL6	TRNL12	16S0D6
2TRNL7	TRNL13	16S0E6
2TRNL7	TRNL14	16S0F6

Template Format:

Run TRNL plates on thermocycler program TRNL_T*:

Step	Temp C	Cycles	Time

Step	Temp C	Cycles	Time
Denature	95	1X	10:00
Denature	95	35X	0:30
Annealing	55	35X	0:30
Extension/Elongation	72	35X	0:30
Hold	4	1X	0:00

MagBead Cleanup:

Equilibrate Beads to room Temperature
Add 15uL of ultra pure water to each well.
Add 24 ul of MagBeads to each well.
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

qPCR

Make 1:1000 dilutions of wells B3 & H6 from the PCR plate by adding 1 ul to 999 ul TE in a deep well plate:

	1	2	3	4	5	6	7	8	9	10	11	12

	10	11	12
A	NTC	NTC	NTC
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2 pM Std	2 pM Std	2 pM Std
G	20 pM Std	20 pM Std	20 pM Std
H

Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	50	500
2 ul	Primer Premix (10X)	50	100
4 ul	Ultra Pure Water	50	200
16 ul	Total Volume	50	800

Add 4 ul of template, pool, or standards to each well:

	1	2	3	4	5	6	7	8	9	10	11	12

	10	11	12
A	NTC	NTC	NTC
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2 pM Std	2 pM Std	2 pM Std
G	20 pM Std	20 pM Std	20 pM Std
H

Genome Technologies Laboratory