1DAVIS

5 plates expected

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)

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)

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

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

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