1RALU

4 plates received 2-12-2025.

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	550	632
5M NaCl	0.12	550	66
1 mg/ml BSA	0.6	550	330
H2O	0.73	550	402
MseI (enzyme)	0.12	550	66
EcoR1 (enzyme)	0.28	550	154
Total	3	550	1650

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	550	550
H2O	0.112	550	61.6
10x T4 Buffer	0.1	550	55
5M NaCl	0.01	550	5.5
1 mg/ml BSA	0.05	550	27.5
T4 DNA ligase	0.1675	550	92.1
Total	1.4	550	770

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:(with 1Spencer)

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

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	300	2856
5x iProof buffer	4	300	1200
10 mM dNTPs	0.4	300	120
50 mM MgCl2	0.4	300	120
5 uM Illumina Primers	1.33	300	399
iProof TAQ	0.2	300	60
DMSO	0.15	300	45
total	16	300	4800

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	300	149	127.5
10 mM dNTPs	0.4	300	140	120
Primers	1.33	300	466	399
Total	2.155	300	755	646.5

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
RALU1	2
RALU2	3
RALU3	4
RALU4	5

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

1RALU

Analytics

Restriction Digestion

Adaptor Ligation

PCR1:(with 1Spencer)

Extra PCR

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