1Spencer

52 samples received 2-6-2025. Extracted via Qiagen Blood and Tissue Kit 2-11-2025. An extraction blank, process blank, 2 lambda controls and a repeat of column 1 will be used to reach a working number.

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 3 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	64	115
5M NaCl	0.12	64	12
1 mg/ml BSA	0.6	64	60
H2O	0.73	64	73
MseI (enzyme)	0.12	64	12
EcoR1 (enzyme)	0.28	64	28
Total	3	64	300

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Reagent	ul/rxn	rxns	ul needed (1.6x)

Reagent	ul/rxn	rxns	ul needed (1.6x)
MseI oligo	1	64	100
H2O	0.112	64	11.2
10x T4 Buffer	0.1	64	10
5M NaCl	0.01	64	1
1 mg/ml BSA	0.05	64	5
T4 DNA ligase	0.1675	64	17
Total	1.4	64	140

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate. Leave out at room temperature for ~2 hours.

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	32	476
5x iProof buffer	4	32	200
10 mM dNTPs	0.4	32	20
50 mM MgCl2	0.4	32	20
5 uM Illumina Primers	1.33	32	66.5
iProof TAQ	0.2	32	10
DMSO	0.15	32	7.5
total	16	32	800

Add 16 ul of PCR1 MM to each well of 2+12(for 4Trout) new hard shell PCR plates with AssistPlus program 16ulMMDisp on 125ul ViaFlo

Add 4uL of template from pooled plates with Benchsmart or Assist Plus PoolWINPlatePCR on 12.5 ul ViaFlo (This pools 4 samples together with each sample being pooled with 2 separate sets of samples)

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C	Cycles	Time

Temp C	Cycles	Time
95*	1X	3:00
98	30X	0:30
60	30X	0:30
72	30X	0:40
72	1X	10:00
4*	1X*	0:00*

*pause step

Extra PCR

Add 2.155 ul of the MM directly to the previous PCR as soon after sample reaches 4C as possible

Reagent	ul/rxn	rxns	ul needed (x 1.6)
5x Iproof buffer	0.425	32	149	21.3
10 mM dNTPs	0.4	32	140	20
Primers	1.33	32	466	66.5
Total	2.155	32	755	108

Then continue with the last four unlisted steps for GBS1 from above:

Temp C	Cycles	Time

Temp C	Cycles	Time
98	1X	3:00
60	1X	2:00
72	1X	10:00
4	1X	0:00

Template	EcoR1 MID plate	Pool Plate	Library	Norm to	Sequencer

Template	EcoR1 MID plate	Pool Plate	Library	Norm to	Sequencer
Spencer1	1

Size Select at 275-375 bp on Pippin Prep 2% no dye gel

Before Size Selection:

250-500 bp selection:

275-375 bp selection:

Genome Technologies Laboratory

1Spencer

Analytics

Restriction Digestion

Adaptor Ligation

PCR1:

Extra PCR

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