Sep 28, 2023

Public workspaceDNA Isolation (Gel Clean-up) V.2

DOI
dx.doi.org/10.17504/protocols.io.81wgbxnk3lpk/v2
  • 1National University of Singapore
NUS iGEM
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DOI: dx.doi.org/10.17504/protocols.io.81wgbxnk3lpk/v2
Protocol Citation: NUS iGEM 2023. DNA Isolation (Gel Clean-up). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxnk3lpk/v2Version created by NUS iGEM
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 28, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 88520
Keywords: DNA Isolation, DNA, Gel, Gel Electrophoresis, Buffer QG, Buffer PE
Abstract
2023 NUS-Singapore iGEM team followed this protocol to isolate the DNA fragments from the agarose gel after the gel electrophoresis.
Protocol materials
ReagentBuffer QGQiagenCatalog #19063
Step 5
ReagentBuffer PEQiagenCatalog #19065
In 3 steps
Safety warnings
  • Proper lab PPE must be worn at all times.
  • When using the LED transilluminator, wear eyewear that is designed to block blue light to protect the eyes.
Prepare and label an Eppondoft tube.
Place the agarose gel onto the LED Transilluminator (blue light) to observe the DNA band(s).
Safety information
  • Wear protective eyewear to protect the eyes from blue light.
  • Turn off the LED transilluminator immediately when it is not in use.

Cut out the target DNA band from the agarose gel.
Put the gel piece into the Eppendorf tube.
Add Amount450 µL of ReagentBuffer QGQiagenCatalog #19063 into the Eppendorf tube.

Heat the Eppendorf tube at Temperature55 °C for Duration00:20:00 in the Thermo-Shaker.

20m
Add Amount150 µL of 100% isopropanol (IPA) into the Eppendorf tube and shake the tube to mix the solution well.

Transfer the whole solution into a QIAquick Spin Column (purple tube with a maximum volume of Amount750 µL ).

Centrifuge it for Centrifigation13 rpm, 00:01:00 .

1m
Discard the flow-through and place the QIAquick column back into the same tube.
Add Amount700 µL of ReagentBuffer PEQiagenCatalog #19065 into the QIAquick column.

Centrifuge it for Centrifigation13 rpm, 00:01:00 .

Discard the flow-through and place the QIAquick column back into the same tube.
Add Amount700 µL of ReagentBuffer PEQiagenCatalog #19065 again into the QIAquick column.

Centrifuge it for Centrifigation13 rpm, 00:01:00 .

Discard the flow-through and place the QIAquick column back into the same tube.
Centrifuge the emptied QIAquick column at Centrifigation13 rpm, 00:01:00 to remove residual ReagentBuffer PEQiagenCatalog #19065 .

1m
Transfer the QIAquick column into the newly labelled Eppendorf tube.
Add Amount30 µL of DI water into the QIAquick column.

Centrifuge the tube at Centrifigation13 rpm, 00:01:00 , ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.

1m
Discard the QIAquick column, the solution left in the Eppendorf tube contains the DNA fragment of interest.
Use the Nanodrop to measure and record the purity and concentration of the DNA fragment.
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU

Keep the isolated DNA fragment in the TemperatureRoom temperature .