Macherey-Nagel Nucleospin 96 Food protocol for bee pollen

Lauren Ponisio, Jocelyn Zorn

Published: 2022-11-03 DOI: 10.17504/protocols.io.kxygxpbrol8j/v1

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Abstract

Steps

UV sterilize supplies for 2 96 well plates worth of extractions: 4 50mL centrifuge tubes, 2 15mL centrifuge tubes, zirconia beads, 2 96 deep well plates and clear strip caps, 2 s-blocks, 2 96 well elution plates, 14 1000uL tip boxes, 2 200uL tip boxes, 2 10uL tip boxes, 6 reagent troughs, and 6 96 well microplates

Aliquot into sterile centrifuge tube and warm in 65°C water bath. You will need to aliquot 30mL per 1/2 plate (48 samples)

Add 540µL to each warmed 30mL

aliquot and invert gently to mix

Add 560µL + solution to each sample

Note

Each sample will receive 550µL Lysis Buffer CF and 10µL Proteinase K

Place in tissue lyser and run at 10Hz for 0h 1m 0s

Note

Make sure pollen ball is removed from bee leg after lysing. If still attached, repeat this step, increasing Hz if needed

Equipment

Value	Label
TissueLyser II	NAME
Bead Mill	TYPE
QIAGEN	BRAND
85300	SKU

Centrifuge 3220x g

Equipment

Value	Label
Eppendorf™ 5810R Centrifuge	NAME
Centrifuge	TYPE
Eppendorf	BRAND
02-262-8187	SKU

Using sterile tweezers, remove bee leg, rinse with 200 proof ethanol, and place leg into labeled sterile microcentrifuge tube. Sterilize tweezers between each use with flame. Leave leg tubes open in fume hood until remaining ethanol has evaporated, then store at -80°C

Note

If extracting pollen not attached to bee leg, skip steps 5-7

Add ~100µL zirconia beads to each pollen sample

Place in tissue lyser and run at 24Hz for 0h 1m 30s , rotate plates 180 degrees, and lyse again at24Hzfor 0h 1m 30s

Equipment

Value	Label
TissueLyser II	NAME
Bead Mill	TYPE
QIAGEN	BRAND
85300	SKU

10.

Place in 65°C water bath for 0h 30m 0s

Note

Incubation time may be increased up to overnight if extraction of DNA from pollen during lysis was not sufficient

11.

Centrifuge 3220x g

12.

Transfer 300µL of supernatant into 96 deep well plate

Note

Samples may be stored at -20°C after this step

13.

Add 300µL (or equal volume)

and 300µL (or equal volume) 200 proof ethanol

Note

Binding Buffer C4 and ethanol may be combined ahead of time. Check note on reagent bottle. If already combined, add 600µL Binding Buffer C4 /EtOH solution. Binding Buffer C4 with EtOH added may be stored at room temperature for up to 1 month

Safety information

Binding Buffer C4 contains guanidine salt - do not mix with bleach

14.

Vortex samples until thoroughly combined

15.

Centrifuge 1500x g,0h 0m 0s for 0h 0m 30s

Note

Do not centrifuge at a higher g-force or for a longer duration - this will precipitate out DNA

16.

Place food binding plate onto s-block and transfer sample to food binding plate. Seal with gas-permeable foil

17.

Centrifuge 3220x g , discard flowthrough

18.

While centrifuging, aliquot out 12mL per plate of and place in 70°C water bath

19.

Add 500µL and seal

Safety information

Wash Buffer CQW contains guanidine salt - do not mix with bleach

20.

Centrifuge 3220x g , discard flowthrough

21.

Add 900µL , do not seal

22.

Centrifuge 3220x g , discard flowthrough

23.

Incubate binding plate 37°C for 0h 30m 0s

24.

Place binding plate over elution block. Add 100µL pre-heated directly onto center of each binding plate filter membrane

25.

Incubate room temperature 0h 5m 0s

26.

Centrifuge 3220x g

27.

Aliquot entirety of product into 3 96 well microplates per DNA plate

28.

Store at -20°C (short term) to -80°C (long term)

Macherey-Nagel Nucleospin 96 Food protocol for bee pollen

Abstract

Steps

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