Sep 10, 2015
QIAgen DNeasy Blood & Tissue kit, cultured cells
- Harold Bien1
- 1Stony Brook University
External link: https://www.qiagen.com/us/resources/resourcedetail?id=6b09dfb8-6319-464d-996c-79e8c7045a50&lang=en
Protocol Citation: Harold Bien 2015. QIAgen DNeasy Blood & Tissue kit, cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dsz6f5
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
Created: September 10, 2015
Last Modified: March 28, 2018
Protocol Integer ID: 1593
Keywords: qiagen dneasy blood, cultured cells qiagen, tissue kit, dneasy blood, cell, tissue, kit
Abstract
QIAgen's DNeasy Blood & Tissue kit (cat#69504 or 69506) for cultured cells
Materials
MATERIALS
1X PBS (Phosphate-buffered saline )
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Ethanol, pureOmnipure Filter CompanyCatalog #4455
Buffer AL, Lysis bufferQiagenCatalog #19076
Buffer AW1, Wash buffer (1), concentrateQiagenCatalog #19081
Buffer AW2, Wash buffer (2), concentrateQiagenCatalog #19072
Buffer AE, Elution buffer, 240mLQiagenCatalog #19077
STEP MATERIALS
1X PBS (Phosphate-buffered saline )
Proteinase K, 2mLQiagenCatalog #19131
Buffer AL, Lysis bufferQiagenCatalog #19076
Ethanol, pureOmnipure Filter CompanyCatalog #4455
Buffer AW1, Wash buffer (1), concentrateQiagenCatalog #19081
Buffer AW2, Wash buffer (2), concentrateQiagenCatalog #19072
Buffer AE, Elution buffer, 240mLQiagenCatalog #19077
1X PBS (Phosphate-buffered saline )
Proteinase K, 2mLQiagenCatalog #19131
Buffer AL, Lysis bufferQiagenCatalog #19076
Ethanol, pureOmnipure Filter CompanyCatalog #4455
Buffer AW1, Wash buffer (1), concentrateQiagenCatalog #19081
Buffer AW2, Wash buffer (2), concentrateQiagenCatalog #19072
Buffer AE, Elution buffer, 240mLQiagenCatalog #19077
Protocol materials
Buffer AL, Lysis bufferQiagenCatalog #19076
Buffer AW1, Wash buffer (1), concentrateQiagenCatalog #19081
1X PBS (Phosphate-buffered saline )
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Buffer AE, Elution buffer, 240mLQiagenCatalog #19077
Ethanol, pureOmnipure Filter CompanyCatalog #4455
Proteinase K, 2mLQiagenCatalog #19131
Buffer AW2, Wash buffer (2), concentrateQiagenCatalog #19072
Troubleshooting
Before start
Preparation of buffer AW1 and AW2
Add ethanol to AW1 and AW2 as directed on bottle. Stable for one year.
If buffer AL has precipitates, warm to 56C until precipitates dissolved
Preheat thermoixer to 56C
Equipment required:
Pipettes and pipette tips
Vortexer
Microcentrifuge tubes, 1.5-1.7mL
Thermomixer for heating at 56C
Centrifuge a maximum of 5 million cells for 5 minutes at 300g
00:05:00
Resuspend pellet in 200μL of PBS
200 µL
1X PBS (Phosphate-buffered saline )
Add 20μL proteinase K.
20 µL
Proteinase K, 2mLQiagenCatalog #19131
Add 200μL Buffer AL and mix thoroughly by vortexing
200 µL
Buffer AL, Lysis bufferQiagenCatalog #19076
Add 200μL pure ethanol and mix by vortexing
200 µL
Ethanol, pureOmnipure Filter CompanyCatalog #4455
Pipet mixture into DNeasy Mini spin column placed on a 2mL collection tube (supplied with kit)
Centrifuge column at ≥6,000g for 1 minute
00:01:00
Discard collection tube and flow through
Place spin column in new 2mL collection tube
Add 500μL buffer AW1
500 µL
Buffer AW1, Wash buffer (1), concentrateQiagenCatalog #19081
Centrifuge for 1 minute at≥6,000g
00:01:00
Discard collection tube and flow through
Add 500μL of Buffer AW2
500 µL
Buffer AW2, Wash buffer (2), concentrateQiagenCatalog #19072
Centrifuge at 20,000g for 3 minutes
00:30:00
Discard collection tube and flow through
Transfer spin column to new (final and labeled) collection tube [optionally sterilized]
Elute DNA by adding 200μL of buffer AE to the center of the spin column
200 µL
Buffer AE, Elution buffer, 240mLQiagenCatalog #19077
Incubate for 1 minute at room temperature
00:01:00
Centrifuge at ≥6,000g for 1 minute
00:01:00
[Optiona]: Repeat step 19 for increased DNA yield