1BEAV

4 full plates plus 7 columns on a 5th. We will add 8 lambda samples as positive controls and to make the partial plate easier to work with the automation. Sequence at AdmeraHealth with Davis2 and 1RALU samples on NovaSeq X. Used SpeedVac to evaporate all liquid from all samples. Resuspended in 25 ul UPW.

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	150	172.5
5M NaCl	0.12	150	18
1 mg/ml BSA	0.6	150	90
H2O	0.73	150	109.5
MseI (enzyme)	0.12	150	18
EcoR1 (enzyme)	0.28	150	42
Total	3	150	450

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	160	160
H2O	0.112	160	18
10x T4 Buffer	0.1	160	16
5M NaCl	0.01	160	1.6
1 mg/ml BSA	0.05	160	8
T4 DNA ligase	0.1675	160	27
Total	1.4	160	224

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	80	761.6
5x iProof buffer	4	80	320
10 mM dNTPs	0.4	80	32
50 mM MgCl2	0.4	80	32
5 uM Illumina Primers	1.33	80	106.4
iProof TAQ	0.2	80	16
DMSO	0.15	80	12
total	16	80	1280

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	80	34
10 mM dNTPs	0.4	80	32
Primers	1.33	80	106.4
Total	2.155	80	172.4

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	Norm to

Template	EcoR1 MID plate	Norm to
DAVIS_BEAV1	2	5
DAVIS_BEAV2	1	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: