RHir1 Hamilton RFS

4-18-2022 Samples have been pooled post-PCR. They still need to be size selected, qPCRed and bio analyzed.

Check In Samples Against List from Jill Hamilton

Samples Arrived 03/31/2022

 Load Submission Data into MISO

Template Plates

Template Plates

RHIR1

RHIR2

RHIR3

RHIR4

RHIR5

RHIR6 (32 samples)

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 7 plates using the 8 channel

Reagent

ul/rxn

rxns

ul needed (1.5x)

10x T4 Buffer

1.15

500

862.5

5M NaCl

0.12

500

90

1 mg/ml BSA

0.6

500

450

H2O

0.73

500

547.5

MseI (enzyme)

0.12

500

90

EcoR1 (enzyme)

0.28

500

210

Total

3

500

2,250

Add 6 ul template to each plate with Benchsmart. Add 6 ul of TE to Wells B4-H4 of RHIR6. 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

500

800

H2O

0.112

500

89.6

10x T4 Buffer

0.1

500

80

5M NaCl

0.01

500

8

1 mg/ml BSA

0.05

500

40

T4 DNA ligase

0.1675

500

134

Total

1.4

500

1120

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Template

EcoR1 MID plate

Template

EcoR1 MID plate

RHIR1

EcoR1 MID plate 1

RHIR2

EcoR1 MID plate 2

RHIR3

EcoR1 MID plate 3

RHIR4

EcoR1 MID plate 4

RHIR5

EcoR1 MID plate 5

RHIR6

EcoR1 MID plate 6

Label reaction plates with MID plate used.

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

Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). Pool C (or C1 - C4) will be recombining plate 5 by quarter plates and Plate 7 by column. Combine columns 5:1-3, 5:4-6, 5:7-9 and 5:10-12 into columns 1-4 of a new plate. Then into columns 5-8 of the same plate add columns 5:1-3, 5:6-4, 5:7-9 and 5:12-10. Into column 9 of this same plate, add columns 1-4 of RHIR6. Into column 10, add columns 1-4 of RHIR6, but flip columns 2 and 4 upside down.

Pool

Plate1

Plate2

Plate3

Plate4

Pool

Plate1

Plate2

Plate3

Plate4

A

RHIR1

RHIR2

RHIR3

RHIR4

Pool

Plate1

Plate2

Plate3

Plate4

Pool

Plate1

Plate2

Plate3

Plate4

B

RHIR1

RHIR2

(FLIPPED)

RHIR3

RHIR4

(FLIPPED)

 

Pool

Plate5 Col 1-3

Plate5 Col 4-6

Plate5 Col 7-9

Plate5 Col 10-12

Pool

Plate5 Col 1-3

Plate5 Col 4-6

Plate5 Col 7-9

Plate5 Col 10-12

C1

RHIR5 (Col 1-3)

RHIR5 (Col 4-6)

RHIR5 (Col 7-9)

RHIR5 (Col 10-12)

 

Pool

Plate5 Col 1-3

Plate5 Col 6-4

Plate5 Col 7-9

Plate5 Col 12-10

Pool

Plate5 Col 1-3

Plate5 Col 6-4

Plate5 Col 7-9

Plate5 Col 12-10

C2

RHIR5 (Col 1-3)

RHIR5 (Col 6-4)

RHIR5 (Col 7-9)

RHIR5 (Col 12-10)

 

Pool

Plate6 Col 1

Plate5 Col 2

Plate5 Col 3

Plate5 Col 4

Pool

Plate6 Col 1

Plate5 Col 2

Plate5 Col 3

Plate5 Col 4

C3

RHIR6 Col1

RHIR6 Col2

RHIR6 Col3

RHIR6 Col4

Pool

Plate6 Col 1

Plate5 Col 2

Plate5 Col 3

Plate5 Col 4

Pool

Plate6 Col 1

Plate5 Col 2

Plate5 Col 3

Plate5 Col 4

C4

RHIR6 Col1

RHIR6 Col2

(FLIPPED)

RHIR6 Col3

RHIR6 Col4

(FLIPPED)

PCR1:

Make MM1 in a 15 ml tube:

Reagent

ul/rxn

rxns

ul needed

Reagent

ul/rxn

rxns

ul needed

H2O

9.52

272

3665.2

5x iProof buffer

4

272

1540

10 mM dNTPs

0.4

272

154

50 mM MgCl2

0.4

272

154

5 uM Illumina Primers

1.33

272

512

iProof TAQ

0.2

272

77

DMSO

0.15

272

57.8

total

16

272

6160

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 2.125 ul of the MM directly to the previous PCR

Reagent

ul/rxn

rxns

ul needed

5x Iproof buffer

0.425

272

185.3

10 mM dNTPs

0.4

272

174.4

Primers

1.33

272

580

Total

2.155

272

939.7

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

Pippin Prep Size Select:

Pool 2 ul from all wells of the 3 final plates into an 8 tube strip. Pool 30 ul from each of these into one tube after mixing via pipette.

Run Final Product on qPCR for check* (Do we still want to do this and the bioanalyzer?)

Result from qPCR check:

HPau_PP_Final:

The full result report can be viewed below:

Mail for sequencing:

  • Fill out the Shipping and Receiving domestic shipping form.

    • correct ORED shipping account number: 10-200-010002-70009-001-6023-0000-0

    • Sequencing Facility Address:

University of Colorado

Brian Woessner

Genomics Core, Bldg RC-2 Room 9400

12700 East 19th Ave.
Aurora, CO 80045

303-724-6050