TRNL Test Samples PCR Test

Test samples were previously extracted.

Make sample spreadsheet and plate test samples by adding 30uL of sample to the corresponding well.
Quantify samples on Synergy HTX.
Normalize samples to 10ng/uL.
Choose 4 plant and 4 stool to run PCR test.
Make 1 step PCR MMX and plate in the template format below (Replace P1, P2, …. and S1, S2… with actual sample names):

PCR MasterMix

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	80	240
0.45	10M dNTPs	80	36
0.3	Kapa HiFi HotStart DNA Pol	80	24
7.25	HPLC H2O	80	580
11	Total Volume	80	880

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.
Primers: _______________ (fill in when primers arrive).

Template Format:

	1	2	3	4	5	6	7	8
A	P1	P2	P3	P4	S1	S2	S3	S4
B	P1	P2	P3	P4	S1	S2	S3	S4
C	P1	P2	P3	P4	S1	S2	S3	S4
D	P1	P2	P3	P4	S1	S2	S3	S4
E	P1	P2	P3	P4	S1	S2	S3	S4
F	P1	P2	P3	P4	S1	S2	S3	S4
G	P1	P2	P3	P4	S1	S2	S3	S4
H	P1	P2	P3	P4	S1	S2	S3	S4

Run on thermocycler program TRNL_T*:

Step	Temp C	Cycles	Time
Denature	95	1X	10:00
Annealing	95	35X	0:30
Annealing** (Row E)	50	35X	0:30
Extension/Elongation	72	35X	2:00
Hold	4	1X	0:00

*TRNL_T program will test the efficiency of the PCR/annealing temperature by running it on a temperature gradient for each row. The table below shows the annealing temperature for each row.

**

Row	Annealing Temperature
A	60
B	57
C	55
D	53
E	50
F	48
G	46
H	44

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

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 TRNL_Test

Make 1:1000 dilutions of column 1,2,3 and 6,7,8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate: (Will fill in with proper sample names.)

	1	2	3	4	5	6	7	8	10	11	12
A	P1_A	P2_A	P3_A	P4_A	S1_A	S2_A	S3_A	S4_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
10 ul	KAPA SYBR FAST qPCR MM (2X)	110	1100
2 ul	Primer Premix (10X)	110	220
4 ul	Ultra Pure Water	110	440
16 ul	Total Volume	110	1760

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

	1	2	3	4	5	6	7	8	10	11	12
A	P1_A	P2_A	P3_A	P4_A	S1_A	S2_A	S3_A	S4_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

Results:

Average results

	1	2	3	4	5	6	7	8
A	P1	P2	P3	P4	S1	S2	S3	S4
B	P1	P2	P3	P4	S1	S2	S3	S4
C	P1	P2	P3	P4	S1	S2	S3	S4
D	P1	P2	P3	P4	S1	S2	S3	S4
E	P1	P2	P3	P4	S1	S2	S3	S4
F	P1	P2	P3	P4	S1	S2	S3	S4
G	P1	P2	P3	P4	S1	S2	S3	S4
H	P1	P2	P3	P4	S1	S2	S3	S4

	1	2	3	4	5	6	7	8
A	P1	P2	P3	P4	S1	S2	S3	S4
B	P1	P2	P3	P4	S1	S2	S3	S4
C	P1	P2	P3	P4	S1	S2	S3	S4
D	P1	P2	P3	P4	S1	S2	S3	S4
E	P1	P2	P3	P4	S1	S2	S3	S4
F	P1	P2	P3	P4	S1	S2	S3	S4
G	P1	P2	P3	P4	S1	S2	S3	S4
H	P1	P2	P3	P4	S1	S2	S3	S4

	1	2	3	4	5	6	7	8
A	P1	P2	P3	P4	S1	S2	S3	S4
B	P1	P2	P3	P4	S1	S2	S3	S4
C	P1	P2	P3	P4	S1	S2	S3	S4
D	P1	P2	P3	P4	S1	S2	S3	S4
E	P1	P2	P3	P4	S1	S2	S3	S4
F	P1	P2	P3	P4	S1	S2	S3	S4
G	P1	P2	P3	P4	S1	S2	S3	S4
H	P1	P2	P3	P4	S1	S2	S3	S4