1TORRES

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	400	460
5M NaCl	0.12	400	48
1 mg/ml BSA	0.6	400	240
H2O	0.73	400	292
MseI (enzyme)	0.12	400	48
EcoR1 (enzyme)	0.28	400	112
Total	3	400	1200

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	450	450
H2O	0.112	450	50.4
10x T4 Buffer	0.1	450	45
5M NaCl	0.01	450	4.5
1 mg/ml BSA	0.05	450	22.5
T4 DNA ligase	0.1675	450	75.4
Total	1.4	450	630

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	1800	17136
5x iProof buffer	4	1800	7200
10 mM dNTPs	0.4	1800	720
50 mM MgCl2	0.4	1800	720
5 uM Illumina Primers	1.33	1800	2394
iProof TAQ	0.2	1800	360
DMSO	0.15	1800	270
total	16	1800	28800

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	1800	765
10 mM dNTPs	0.4	1800	720
Primers	1.33	1800	2394
Total	2.155	1800	3879

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
AR1	5	1+2	1	10	Nova
AR2	6	1+2	1	10	Nova
AR3	7	3+4	1	10	Nova
AR4	8	3+4	1	10	Nova

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