Skip to end of metadata
Go to start of metadata

You are viewing an old version of this page. View the current version.

Compare with Current View Page History

« Previous Version 4 Current »

Received New DNAs 9-6-2022

Digests started 9-12-2022

Ligation started 9-14-2022

 

1

2

3

4

5

6

7

8

9

10

11

12

A

L3_PJD83

L6_PJD86

L1_PJD94

L2_PJD91

B

H6_PJD98

H2_PJD95

L7_PJD97

I10_PJD75

C

B2377_PJD57

H5_PJD82

I7_PJD80

H4_PJD89

D

L9_PJD99

I1_PJD73

L4_PJD87

I14_PJD81

E

B2461_PJD60

L5_PJD77

I9_PJD74

I4_PJD85

F

B2437_PJD58

I13_PJD92

I11_PJD90

I5_PJD79

G

I12_PJD96

I6_PJD88

I2_PJD84

H1_PJD76

H

B2453_PJD59

I15_PJD78

L10_PJD61

H3_PJD93

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 2 plates using the 8 channel

Reagent

ul/rxn

rxns

ul needed (1.5x)

10x T4 Buffer

1.15

32

57.5

5M NaCl

0.12

32

6

1 mg/ml BSA

0.6

32

30

H2O

0.73

32

36.5

MseI (enzyme)

0.12

32

6

EcoR1 (enzyme)

0.28

32

14

Total

3

32

150

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

32

52

H2O

0.112

32

5.8

10x T4 Buffer

0.1

32

5.2

5M NaCl

0.01

32

0.52

1 mg/ml BSA

0.05

32

2.6

T4 DNA ligase

0.1675

32

8.7

Total

1.4

32

72.8

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

Template

EcoR1 MID plate

1BUN2 (1PJD2)

EcoR1 MID plate 4

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. Load Pool A1 into columns 1-6 and A2 into columns 7-12 of the pooling plate. 

Pool

Plate1 Col 1

Plate1 Col 2

Plate1 Col 3

Plate2 Col 4

 

Pool

Plate1 Col 1

Plate1 Col 2 (flipped)

Plate1 Col 3

Plate2 Col 4(flipped)

 

PCR1:

Make MM1 in a 1.5mL tube:

Reagent

ul/rxn

rxns (x1.3)

ul needed

H2O

9.52

16

238

5x iProof buffer

4

16

100

10 mM dNTPs

0.4

16

10

50 mM MgCl2

0.4

16

10

5 uM Illumina Primers

1.33

16

33.25

iProof TAQ

0.2

16

5

DMSO

0.15

16

3.75

total

16

16

400

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

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 (x1.6)

ul needed

5x Iproof buffer

0.425

16

10.2

10 mM dNTPs

0.4

16

9.6

Primers

1.33

16

31.9

Total

2.155

16

51.7

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

Bioanalyzer:

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 on Bioanalyzer or Tape Station. Email results to Josh for size selection.

Pippin Prep Size Select:

Run Final Product on qPCR for check

Result from qPCR check:

The full result report can be viewed below:

Mail for sequencing:

  • No labels