Versions Compared

Old Version 2

changes.mady.by.user Shannon Harris

Saved on

compared with

New Version 3

changes.mady.by.user Gregg Randolph

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

Test Plates

  • BS_PLT1_Norm (4 samples)

  • BS_PLT2_Norm (4 samples)

PCR MasterMix

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

80

240

0.45

10M dNTPs

80

36

0.3

Kapa HiFi HotStart DNA Pol

80

24

7.25

HPLC H2O

80

580

11

Total Volume

80

880

  • Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.

  • Primers: _______________ (fill in when primers arrive).

Template Format:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

B

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

C

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

D

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

E

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

F

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

G

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

H

S1

BSP1 C1

S2

BSP1 C1

S3

BSP1 C1

S4

BSP1 C1

S5

BSP1 C1

S6

BSP1 C1

S7

BSP1 C1

S8

BSP1 C1

 

 

 

 

Run on thermocycler program BSP*:

Step

Temp C

Cycles

Time

Denature

95

1X

10:00

Annealing

94

35X

0:30

Annealing** (Row E)

55

35X

0:30

Extension/Elongation

72

35X

1:00

Extension/Elongation

74

1X

9:00

Hold

4

1X

0:00

*BSP program will test the efficiency of the PCR/annealing temperature by running it on a temperature gradient for each row. The table below shows the annealing temperature for each row.

**

Row

Annealing Temperature

A

63

B

61

C

59

D

57

E

55

F

53

G

50

H

48

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

...

  • Make 1:1000 dilutions of column 1,2,3 and 6,7,8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate: (Will fill in with proper sample names.)

 

1

2

3

4

5

6

7

8

9

10

11

12

A

S1_A

S2_A

S3_A

S4_A

S5_A

S6_A

S7_A

S8_A

 

NTC

NTC

NTC

B

 

 

 

 

 

 

 

 

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

 

 

 

 

 

 

 

 

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

 

 

 

 

 

 

 

 

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

 

 

 

 

 

 

 

 

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

 

 

 

 

 

 

 

 

 

2 pM Std

2 pM Std

2 pM Std

G

 

 

 

 

 

 

 

 

 

20 pM Std

20 pM Std

20 pM Std

H

 

 

 

 

 

 

 

 

 

 

 

 

  • Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn

Reagent

# of rxns

ul needed

10 ul

KAPA SYBR FAST qPCR MM (2X)

110

1100

2 ul

Primer Premix (10X)

110

220

4 ul

Ultra Pure Water

110

440

16 ul

Total Volume

110

1760

  • Add 4 ul of template, pool, or standards to each well:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

S1_A

S2_A

S3_A

S4_A

S5_A

S6_A

S7_A

S8_A

 

NTC

NTC

NTC

B

 

 

 

 

 

 

 

 

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

 

 

 

 

 

 

 

 

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

 

 

 

 

 

 

 

 

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

 

 

 

 

 

 

 

 

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

 

 

 

 

 

 

 

 

 

2 pM Std

2 pM Std

2 pM Std

G

 

 

 

 

 

 

 

 

 

20 pM Std

20 pM Std

20 pM Std

H

 

 

 

 

 

 

 

 

 

 

 

 

Results:

Average results