BrockPna

Owned by Gregg Randolph
Last updated: Jul 24, 2024
3 min read

16S V4 Project with PNA blockers

Dilute Stock PNAs to working concentration

50 uM PNA blockers mPNA and pPNA

6 uM allows for 5 ul per combined PNA to reach 2 uM and 1.25 ul for 0.5 uM

12 ul each PNA in 76 ul TE makes 100 ul 6uM PNAs with both m and p

Dilute DNAs to 10 ng/ul

Use concentrations provided

MasterMix for standard

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

150

450

0.45

10M dNTPs

150

68

0.3

Kapa HiFi HotStart DNA Pol

150

45

5.25

HPLC H2O

150

787

9

Total Volume

150

1350

  • Add 9 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of each of the primers).

MasterMix for PNA work

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

50

150

0.45

10M dNTPs

50

22.5

0.3

Kapa HiFi HotStart DNA Pol

50

15

2.25

HPLC H2O

50

112.5

6

Total Volume

50

300

  • Add 6 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. ( one needs 2 ul of template and 2 ul of the primers).

PNA volumes

 

 

 

 

 

 

 

 

1.25

0

1.25

0

1.25

2.5

1.25

2.5

1.25

5

1.25

5

1.25

0

1.25

0

1.25

2.5

1.25

2.5

1.25

5

1.25

5

1.25

 

1.25

 

1.25

 

1.25

 

Water volumes

1

2

3

4

1

2

3

4

3.75

5

3.75

5

3.75

2.5

3.75

2.5

3.75

0

3.75

0

3.75

5

3.75

5

3.75

2.5

3.75

2.5

3.75

0

3.75

0

3.75

 

3.75

 

3.75

 

3.75

 

PCR

Run STD on Thermocycler Program GSAF36:

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00*

98

36X

0:30

62

36X

0:30

72

36X

0:30

72

1X

5:00

4

1X

0:00

Run PNA on PNA_GSAF

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00*

98

36X

0:30

78

36X

0:15

62

36X

0:30

72

36X

0:30

72

1X

5:00

4

1X

0:00

MagBead Cleanup:

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

Manually, it was done:

  • Equilibrate Beads to room Temperature

  • Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

  • Pipette mix up and down 10 times.

  • Incubate at RT for 5 minutes

  • Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

  • Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

  • Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

  • Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

  • Reaspirate from each well to assure maximum EtOH removal

  • Allow plate to air dry for 7 minutes.

  • Remove sample plate from magnet plate.

  • Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

  • Place sample plate back on magnet for 5 minutes or until all wells are cleared.

  • Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

  • Store in -20 until ready to qPCR

TapeStation

Pool:

page3image9915856

Samples:

Pool, followed by Brock 2, column 2 (sample 5 is the blank)

page1image11215808

Blank:

qPCR

45 nM

2.2 ul sample plus 97.8 ul RSB to make 1 nM

 

Sequence Dilution

Dilute to 1 nM:

2.2 ul sample plus 97.8 ul RSB

Add 10% PhiX and dilute to 65 pM:

13 ul sample plus 1.3 ul PhiX plus 185.7 RSB