Sep 28, 2023

Public workspacePCR Clean-up

DOI
dx.doi.org/10.17504/protocols.io.ewov1q212gr2/v1
  • 1National University of Singapore
NUS iGEM
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov1q212gr2/v1
Protocol Citation: NUS iGEM 2023. PCR Clean-up. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q212gr2/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 28, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 88521
Keywords: PCR Clean-up, PCR, DNA, DNA Isolation
Abstract
2023 NUS-Singapore iGEM team followed this protocol to isolate the known DNA fragments from the PCR product without running the gel electrophoresis.
Protocol materials
ReagentPB bufferQiagenCatalog #19066
Step 1
ReagentBuffer PEQiagenCatalog #19065
In 3 steps
Safety warnings
Proper lab PPE must be worn at all times.
Add 5 times the sample's volume of ReagentPB bufferQiagenCatalog #19066 into the PCR tube with the PCR product.

Transfer the whole solution into a QIAquick Spin Column (purple tube) and ensure that the sample is dripped onto the white membrane in the column.
Centrifuge the tube at 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 at 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 at 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 it 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 fragments.
Use the Nanodrop to measure and record the purity and concentration of the DNA fragments.
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU