Nov 02, 2022
Macherey-Nagel Nucleospin 96 Food protocol for bee pollen
- 1University of Oregon
Protocol Citation: Lauren Ponisio, Jocelyn Zorn 2022. Macherey-Nagel Nucleospin 96 Food protocol for bee pollen. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxpbrol8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2021
Last Modified: November 02, 2022
Protocol Integer ID: 52980
Funders Acknowledgement:
Foundation for Food and Agricultural Research
Grant ID: CA18-SS-0000000009
Abstract
Macherey-Nagel Nucleospin 96 Food protocol for bee pollen
Protocol materials
Wash Buffer C5Macherey-NagalCatalog #740931
Step 21
Lysis Buffer CFMacherey-NagalCatalog #740946
In 3 steps
Proteinase KMacherey-NagalCatalog #740506
In 2 steps
Binding Buffer C4Macherey-NagalCatalog #740366.250
Step 13
Elution Buffer CEMacherey-Nagal
In 2 steps
Wash Buffer CQWMacherey-NagalCatalog #740313.125
Step 19
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 Lysis Buffer CFMacherey and NagelCatalog #740946 into sterile centrifuge tube and warm in 65 °C water bath. You will need to aliquot 30 mL per 1/2 plate (48 samples)
Add 540 µL Proteinase KMacherey and NagelCatalog #740506 to each warmed 30 mL
Lysis Buffer CFMacherey and NagelCatalog #740946 aliquot and invert gently to mix
Add 560 µL Lysis Buffer CFMacherey and NagelCatalog #740946 + Proteinase KMacherey and NagelCatalog #740506 solution to each sample
Note
Each sample will receive 550 µL Lysis Buffer CFMacherey NagelCatalog #740946
and 10 µL Proteinase KMacherey NagelCatalog #740506
Place in tissue lyser and run at 10 Hz for 00:01:00
Note
Make sure pollen ball is removed from bee leg after lysing. If still attached, repeat this step, increasing Hz if needed
Equipment
TissueLyser II
NAME
Bead Mill
TYPE
QIAGEN
BRAND
85300
SKU
LINK
1m
Centrifuge 3220 x g, 00:01:00
Equipment
Eppendorf™ 5810R Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
02-262-8187
SKU
LINK
1m
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 ~100 µL zirconia beads to each pollen sample
Place in tissue lyser and run at 24 Hz for 00:01:30 , rotate plates 180 degrees, and lyse again at24 Hz for 00:01:30
Equipment
TissueLyser II
NAME
Bead Mill
TYPE
QIAGEN
BRAND
85300
SKU
LINK
3m
Place in 65 °C water bath for 00:30:00
Note
Incubation time may be increased up to overnight if extraction of DNA from pollen during lysis was not sufficient
30m
Centrifuge 3220 x g, 00:35:00
35m
Transfer 300 µL of supernatant into 96 deep well plate
Note
Samples may be stored at -20 °C after this step
Add 300 µL (or equal volume) Binding Buffer C4Macherey and NagelCatalog #740366.250
and 300 µL (or equal volume) 200 proof ethanol
Note
Binding Buffer C4Macherey NagelCatalog #740366.250 and ethanol may be combined ahead of time. Check note on reagent bottle. If already combined, add 600 µL Binding Buffer C4Macherey NagelCatalog #740366.250 /EtOH solution. Binding Buffer C4Macherey NagelCatalog #740366.250 with EtOH added may be stored at room temperature for up to 1 month
Safety information
Binding Buffer C4Macherey NagelCatalog #740366.250 contains guanidine salt - do not mix with bleach
Vortex samples until thoroughly combined
Centrifuge 1500 x g for 00:00:30
Note
Do not centrifuge at a higher g-force or for a longer duration - this will precipitate out DNA
30s
Place food binding plate onto s-block and transfer sample to food binding plate. Seal with gas-permeable foil
Centrifuge 3220 x g, 00:09:00 , discard flowthrough
9m
While centrifuging, aliquot out 12 mL per plate of Elution Buffer CEMacherey and Nagel and place in 70 °C water bath
Add 500 µL Wash Buffer CQWMacherey and NagelCatalog #740313.125 and seal
Safety information
Wash Buffer CQWMacherey NagelCatalog #740313.125 contains guanidine salt - do not mix with bleach
Centrifuge 3220 x g, 00:04:00 , discard flowthrough
4m
Add 900 µL Wash Buffer C5Macherey and NagelCatalog #740931 , do not seal
Centrifuge 3220 x g, 00:20:00 , discard flowthrough
20m
Incubate binding plate 37 °C for 00:30:00
30m
Place binding plate over elution block. Add 100 µL pre-heated Elution Buffer CEMacherey and Nagel directly onto center of each binding plate filter membrane
Incubate room temperature 00:05:00
5m
Centrifuge 3220 x g, 00:04:00
4m
Aliquot entirety of product into 3 96 well microplates per DNA plate
Store at -20 °C (short term) to -80 °C (long term)