2DAVIS

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. Hold off on sequencing unless instructed to proceed!

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	700	805
5M NaCl	0.12	700	84
1 mg/ml BSA	0.6	700	420
H2O	0.73	700	511
MseI (enzyme)	0.12	700	84
EcoR1 (enzyme)	0.28	700	196
Total	3	700	2100

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)
MseI oligo	1	700	700
H2O	0.112	700	78.4
10x T4 Buffer	0.1	700	70
5M NaCl	0.01	700	7
1 mg/ml BSA	0.05	700	35
T4 DNA ligase	0.1675	700	117.25
Total	1.4	700	980

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)
H2O	9.52	350	3332
5x iProof buffer	4	350	1400
10 mM dNTPs	0.4	350	140
50 mM MgCl2	0.4	350	140
5 uM Illumina Primers	1.33	350	465.5
iProof TAQ	0.2	350	70
DMSO	0.15	350	52.5
total	16	350	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
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	350	149
10 mM dNTPs	0.4	350	140
Primers	1.33	350	466
Total	2.155	350	755

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

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
PSMA1
PSMA2
PSMA3
PSMA4*
ANBO1

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