Wagner Trout 5 (5Trout)

Setup Notes

9 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

  • Cleanup each full pool via ultra purification

  • Size select for 300-366bp fragments size window via Pippin Prep

  • Mix of on campus and Sending Out for Sequencing

  • 5% PhiX spike

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Pooling Notes

Library 1: MPR1; MPR2; YCT6

 

Library 2: MPR3; MPR5; GYCT3

 

Library 3: MPR4; MPR6; GYCT7

Quantify and normalize samples:

  • Normalize plate reader with TE for all plates

  • Quantify all plates

  • Normalize to 30 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 9 plates using the 8 channel

Reagent

ul/rxn

rxns

ul needed (1.5x)

10x T4 Buffer

1.15

860

989

5M NaCl

0.12

860

103.2

1 mg/ml BSA

0.6

860

516

H2O

0.73

860

628

MseI (enzyme)

0.12

860

103.2

EcoR1 (enzyme)

0.28

860

241

Total

3

860

2580

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 of the 7 new plates and the plates from 1BROSE.

Reagent

ul/rxn

rxns

ul needed (1.6x)

Reagent

ul/rxn

rxns

ul needed (1.6x)

MseI oligo

1

1400

1400

H2O

0.112

1400

156.8

10x T4 Buffer

0.1

1400

140

5M NaCl

0.01

1400

14

1 mg/ml BSA

0.05

1400

70

T4 DNA ligase

0.1675

1400

234.5

Total

1.4

1400

1960

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

GTL Plate

EcoR1 MID plate

Pool Plate

Library

Norm to

Sequencer

GTL Plate

EcoR1 MID plate

Pool Plate

Library

Norm to

Sequencer

MPR1

1

 

1

30

Next

MPR2

2

 

1

30

Next

YCT6

3

 

1

30

Next

MPR3

1

 

2

30

Next

MPR5

2

 

2

30

Next

GYTC3

7 or 8

 

2

30

Next

MPR4

1

 

3

30

Next

MPR6

2

 

3

30

Next

GYTC7

8 or 7

 

3

30

Next

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

Add 68 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

 

 PCR1:

Reagent

ul/rxn

rxns

ul needed (x1.4)

 

Reagent

ul/rxn

rxns

ul needed (x1.4)

 

H2O

9.52

350

3094

4760

5x iProof buffer

4

350

1300

2000

10 mM dNTPs

0.4

350

130

200

50 mM MgCl2

0.4

350

130

200

5 uM Illumina Primers

1.33

350

432

665

iProof TAQ

0.2

350

65

100

DMSO

0.15

350

49

75

total

16

350

5200

8000

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

360

230

10 mM dNTPs

0.4

360

92

Primers

1.33

360

306

Total

2.155

360

496

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

Pool Using PCR Plates with Assist Plus

Organize plates by Library # above, vortex, and quick spin

Use 4GbsPoolx8

Use single channel to combine 48 ul from each well of a column into a separate labeled tube.

Pippin Prep Size Select (300-366 bp select):

 First attempt showed too little product. I will concentrate via speedvac and cleanup concentration via Nucleofast 96 PCR.

Run Final Products on Tapestation for size check

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Run Final Product on qPCR for check and quant

Result from qPCR check :

 

Sample Name

qPCR

Sample ul

RSB ul

Sample Name

qPCR

Sample ul

RSB ul

Exp5Trout1

1.86 nM

53.8

46.2

5Trout1Cleaned

9.02 nM

11.1

88.9

5Trout1Org

3.05 nM

32.8

67.2

load =800 pM

80 ul 1 nM stock, 4 ul 1 nM PhiX, 16 ul RSB