1ABAX (WY Toad Project)

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

480

690

5M NaCl

0.12

480

72

1 mg/ml BSA

0.6

480

360

H2O

0.73

480

438

MseI (enzyme)

0.12

480

72

EcoR1 (enzyme)

0.28

480

168

Total

3

480

 1800

Tubes:

Reagent

ul/rxn

rxns

ul needed (1.5x)

10x T4 Buffer

1.15

56

69

5M NaCl

0.12

56

7.2

1 mg/ml BSA

0.6

56

36

H2O

0.73

56

43.8

MseI (enzyme)

0.12

56

7.2

EcoR1 (enzyme)

0.28

56

16.8

Total

3

56

 180

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

536

661

H2O

0.112

536

74

10x T4 Buffer

0.1

536

66.1

5M NaCl

0.01

536

6.61

1 mg/ml BSA

0.05

536

33

T4 DNA ligase

0.1675

536

72.7

Total

1.4

536

 110.72

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.

PCR1:

Reagent

ul/rxn

rxns

ul needed (x1.4)

Reagent

ul/rxn

rxns

ul needed (x1.4)

H2O

9.52

268

2761

5x iProof buffer

4

268

1160

10 mM dNTPs

0.4

268

116

50 mM MgCl2

0.4

268

116

5 uM Illumina Primers

1.33

268

385

iProof TAQ

0.2

268

58

DMSO

0.15

268

43.5

total

16

268

4640

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

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

Reagent

ul/rxn

rxns

ul needed (x 1.6)

5x Iproof buffer

0.425

272

136

10 mM dNTPs

0.4

272

128

Primers

1.33

272

426

Total

2.155

272

690

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

Size Select at 275-375bp

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